Search

Epidermal growth factor receptor (EGFR) is involved in development and progression of many human cancers. We have previously demonstrated that the ubiquitin-specific peptidase Usp18 (Ubp43) is a potent regulator of EGFR protein expression. Here we report that the 3'-untranslated region (3'-UTR) of the EGFR message modulates RNA translation following cell treatment with Usp18 siRNA, suggesting microRNA as a possible mediator. Given earlier evidence of EGFR regulation by the microRNA miR-7, we assessed whether miR-7 mediates Usp18 siRNA effects. We found that Usp18 depletion elevates miR-7 levels in several cancer cell lines because of a transcriptional activation and/or mRNA stabilization of miR-7 host genes and that miR-7 acts downstream of Usp18 to regulate EGFR mRNA translation via the 3'-UTR. Also, depletion of Usp18 led to a decrease in protein levels of other known oncogenic targets of miR-7, reduced cell proliferation and soft agar colony formation, and increased apoptosis. Notably, all of these phenotypes were reversed by a specific inhibitor of miR-7. Thus, our findings support a model in which Usp18 inhibition promotes up-regulation of miR-7, which in turn inhibits EGFR expression and the tumorigenic activity of cancer cells.

Emerging studies have identified microRNAs (miRNAs) as possible therapeutic tools for the treatment of glioma, the most aggressive brain tumor. Their important targets in this tumor are not well understood. We recently found that the Notch pathway is a target of miRNA-326. Ectopic expression of miRNA-326 in glioma and glioma stem cells induced their apoptosis and reduced their metabolic activity. Computational target gene prediction revealed pyruvate kinase type M2 (PKM2) as another target of miRNA-326. PKM2 has recently been shown to play a key role in cancer cell metabolism. To investigate whether it might be a functionally important target of miR-326, we used RNA interference to knockdown PKM2 expression in glioma cells. Transfection of the established glioma and glioma stem cells with PKM2 siRNA reduced their growth, cellular invasion, metabolic activity, ATP and glutathione levels, and activated AMP-activated protein kinase. The cytotoxic effects exhibited by PKM2 knockdown in glioma and glioma stem cells were not observed in transformed human astrocytes. Western blot analysis of human glioblastoma specimens showed high levels of PKM2 protein, but none was observed in normal brain samples. Strikingly, cells with high levels of PKM2 expressed lower levels of miR-326, suggestive of endogenous regulation of PKM2 by miR-326. Our data suggest PKM2 inhibition as a therapy for glioblastoma, with the potential for minimal toxicity to the brain.

Little is known of microRNA interactions with cellular pathways. Few reports have associated microRNAs with the Notch pathway, which plays key roles in nervous system development and in brain tumors. We previously implicated the Notch pathway in gliomas, the most common and aggressive brain tumors. While investigating Notch mediators, we noted microRNA-326 was upregulated following Notch-1 knockdown. This neuronally expressed microRNA was not only suppressed by Notch but also inhibited Notch proteins and activity, indicating a feedback loop. MicroRNA-326 was downregulated in gliomas via decreased expression of its host gene. Transfection of microRNA-326 into both established and stem cell-like glioma lines was cytotoxic, and rescue was obtained with Notch restoration. Furthermore, miR-326 transfection reduced glioma cell tumorigenicity in vivo. Additionally, we found microRNA-326 partially mediated the toxic effects of Notch knockdown. This work demonstrates a microRNA-326/Notch axis, shedding light on the biology of Notch and suggesting microRNA-326 delivery as a therapy.

microRNAs are noncoding RNAs inhibiting expression of numerous target genes, and a few have been shown to act as oncogenes or tumor suppressors. We show that microRNA-7 (miR-7) is a potential tumor suppressor in glioblastoma targeting critical cancer pathways. miR-7 potently suppressed epidermal growth factor receptor expression, and furthermore it independently inhibited the Akt pathway via targeting upstream regulators. miR-7 expression was down-regulated in glioblastoma versus surrounding brain, with a mechanism involving impaired processing. Importantly, transfection with miR-7 decreased viability and invasiveness of primary glioblastoma lines. This study establishes miR-7 as a regulator of major cancer pathways and suggests that it has therapeutic potential for glioblastoma.