Background

Results

Conclusion

Glioblastoma is the most common primary malignant tumor of the central nervous system, and is usually treated with maximal surgical resection followed by radiotherapy and concurrent and adjuvant temozolomide. Despite treatment, the tumor almost always recurs, carrying significant mortality, but the specific biological mechanisms by which it recurs are poorly characterized, though it has been suggested that radiotherapy may play a role. microRNA-34c has been shown to be increased by DNA damage in a nuclear ABL-specific manner, and has increased abundance in bystander effect-inducing extracellular vesicles from irradiated cells. We sought to understand whether ionizing radiation could induce expression and secretion of miR-34c in glioblastoma, and if not, whether exogenous miR-34c could induce bystander effects. We exposed validated p53-wildtype and p53-null glioblastoma cell lines to ionizing radiation and found that miR-34c induction did not occur in glioblastoma and that exposing radiation-naïve glioblastoma responder cells to conditioned media from irradiated glioblastoma producer cells did not induce expression of miR-34c in bystander cells. Using HEK-293T cells as extracellular vesicle-producing cells following transfection with miR-34c minigenes generated conditioned media that was capable of inducing ROS and γH2AX in glioblastoma responder cells. MicroRNA mimics were also used to directly transfect glioblastoma cells, and increased the level of nuclear foci of a 53BP1-mCherry fusion protein in a stable glioma line.

Non-genomic estrogen (E2) signaling has previously been implicated in promoting E2-dependent proliferation, but the mechanism by which this occurs is not known. Here, we found a requirement for the protein tyrosine kinase, SRC, in cell cycle progression and proliferation in quiescent estrogen receptor (ER)-positive breast cancer cells stimulated with E2 in vitro. Using a SRC family kinase (SFK) inhibitor, SU11333, and shRNA mediated knockdown, we demonstrated that SRC, but not YES, is required for cell cycle progression and proliferation in vitro. Using the SRC/ABL selective kinase inhibitor, saracatinib, we also demonstrated that SFKs were required for tumor growth in vivo in an orthotopic xenograft model. Expression of MYC is required for E2-dependent proliferation, and MYC mRNA levels are induced by SRC by stabilization of MYC mRNA rather than activation of transcription. This stabilization required the RNA-binding protein N-IMP1 which we found to be expressed in several ER-positive breast cancer cell lines. N-IMP1 stabilization of MYC mRNA required SRC phosphorylation of Y260.

We also observed a role for SRC in overcoming a p53 block to cell cycle progression after E2 stimulation. Loss of p53 in MCF7 cells abrogated a need for SRC in promoting cell cycle progression and proliferation in vitro. In an in vivo MCF7 orthotopic xenograft model, we found that saracatinib inhibited growth of tumors regardless of p53 expression. Using shRNA-mediated knockdown of SRC, we also demonstrated that SRC was required for tumor grafting in mice. We also used an inducible shRNA system to knockdown SRC in established tumors. Here, we show SRC inhibition combined with loss of p53 led to static tumors whereas SRC inhibition in the presence of p53 leads to tumor regression. Loss of p53 led to an increase in MYC mRNA due to increased basal mRNA stabilization and an increase in N-IMP1 protein levels. Loss of p53 also abrogated the ability of E2 to stimulate MYC mRNA stabilization. Lastly, a defect in proliferation was observed in MCF7 cells overexpressing mutations of known SRC target tyrosines in MDM2 (Y281F/Y302F) suggesting a possibility for MDM2 as mediator of SRC inhibition of p53 function.