Extracellular vesicles transfer nuclear Abl-dependent and radiation-induced miR-34c into unirradiated cells to cause bystander effects.

Ionizing radiation (IR) not only activates DNA damage response (DDR) in irradiated cells but also induces bystander effects (BE) in cells not directly targeted by radiation. How DDR pathways activated in irradiated cells stimulate BE is not well understood. We show here that extracellular vesicles secreted by irradiated cells (EV-IR), but not those from unirradiated controls (EV-C), inhibit colony formation in unirradiated cells by inducing reactive oxygen species (ROS). We found that µEV-IR from Abl nuclear localization signal-mutated ( Abl-µNLS) cells could not induce ROS, but expression of wild-type Abl restored that activity. Because nuclear Abl stimulates miR-34c biogenesis, we measured miR-34c in EV and found that its levels correlated with the ROS-inducing activity of EV. We then showed that EV from miR-34c minigene-transfected, but unirradiated cells induced ROS; and transfection with miR-34c-mimic, without radiation or EV addition, also induced ROS. Furthermore, EV-IR from miR34-family triple-knockout cells could not induce ROS, whereas EV-IR from wild-type cells could cause miR-34c increase and ROS induction in the miR-34 triple-knockout cells. These results establish a novel role for extracellular vesicles in transferring nuclear Abl-dependent and radiation-induced miR-34c into unirradiated cells to cause bystander oxidative stress

Nuclear ABL Programs Extracellular Vesicles for Transmission of Ionizing Radiation-Induced Bystander Effects

Radiation therapy is used to treat many diseases such as cancer. However, off-targeted side-effects called bystander effects are observed in patients post radiotherapy. Bystander effects are when non-irradiated cells exhibit similar effects as directly irradiated cells. Understanding how these bystander effects are mediated can help improve cancer treatment. Previous work from our lab has shown that extracellular vesicles derived from conditioned media of irradiated mouse embryonic fibroblasts mediate radiation-induced bystander effects by inhibiting clonogenic survival, increase γH2AX, and increase ROS. However, when the protein Abl is mutated so that it no longer can enter the nucleus, the EVs from the conditioned media of these irradiated cells do not cause bystander effects. When nuclear Abl is reconstituted into these mutant cells, the EVs from the conditioned media of these irradiated cells cause bystander effects such as increase γH2AX and increase ROS. This suggests that nuclear Abl is required for extracellular vesicle mediated radiation-induced bystander effects. Abl inhibitors are FDA approved and might be viable treatments to reduce bystander effects in patients undergoing radiation therapy. Additionally, in a paper previously published by our lab, nuclear Abl is required for miR-34c processing. Since EVs can contain miRNAs as cargo, miR-34c could be involved in the nuclear Abl dependent extracellular vesicle mediated radiation-induced bystander effects. This offers another possible treatment target to counter radiation-induced bystander effects

Extracellular vesicles transfer nuclear Abl-dependent and radiation-induced miR-34c into unirradiated cells to cause bystander effects.

Ionizing radiation (IR) not only activates DNA damage response (DDR) in irradiated cells but also induces bystander effects (BE) in cells not directly targeted by radiation. How DDR pathways activated in irradiated cells stimulate BE is not well understood. We show here that extracellular vesicles secreted by irradiated cells (EV-IR), but not those from unirradiated controls (EV-C), inhibit colony formation in unirradiated cells by inducing reactive oxygen species (ROS). We found that µEV-IR from Abl nuclear localization signal-mutated ( Abl-µNLS) cells could not induce ROS, but expression of wild-type Abl restored that activity. Because nuclear Abl stimulates miR-34c biogenesis, we measured miR-34c in EV and found that its levels correlated with the ROS-inducing activity of EV. We then showed that EV from miR-34c minigene-transfected, but unirradiated cells induced ROS; and transfection with miR-34c-mimic, without radiation or EV addition, also induced ROS. Furthermore, EV-IR from miR34-family triple-knockout cells could not induce ROS, whereas EV-IR from wild-type cells could cause miR-34c increase and ROS induction in the miR-34 triple-knockout cells. These results establish a novel role for extracellular vesicles in transferring nuclear Abl-dependent and radiation-induced miR-34c into unirradiated cells to cause bystander oxidative stress