Benjamin Kefas

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.

MicroRNA-34a (miR-34a) is a transcriptional target of p53 that is down-regulated in some cancer cell lines. We studied the expression, targets, and functional effects of miR-34a in brain tumor cells and human gliomas. Transfection of miR-34a down-regulated c-Met in human glioma and medulloblastoma cells and Notch-1, Notch-2, and CDK6 protein expressions in glioma cells. miR-34a expression inhibited c-Met reporter activities in glioma and medulloblastoma cells and Notch-1 and Notch-2 3'-untranslated region reporter activities in glioma cells and stem cells. Analysis of human specimens showed that miR-34a expression is down-regulated in glioblastoma tissues as compared with normal brain and in mutant p53 gliomas as compared with wild-type p53 gliomas. miR-34a levels in human gliomas inversely correlated to c-Met levels measured in the same tumors. Transient transfection of miR-34a into glioma and medulloblastoma cell lines strongly inhibited cell proliferation, cell cycle progression, cell survival, and cell invasion, but transfection of miR-34a into human astrocytes did not affect cell survival and cell cycle status. Forced expression of c-Met or Notch-1/Notch-2 transcripts lacking the 3'-untranslated region sequences partially reversed the effects of miR-34a on cell cycle arrest and cell death in glioma cells and stem cells, respectively. Also, transient expression of miR-34a in glioblastoma cells strongly inhibited in vivo glioma xenograft growth. Together, these findings represent the first comprehensive analysis of the role of miR-34a in gliomas. They show that miR-34a suppresses brain tumor growth by targeting c-Met and Notch. The results also suggest that miR-34a could serve as a potential therapeutic agent for brain tumors.

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.

The aim of this work was to study the effect of a sustained activation of AMP-activated protein kinase (AMPK) on liver cell survival. AMPK activation was achieved by incubating FTO2B cells with AICA-riboside, which is transformed into ZMP, an AMP analogue, or by adenoviral transfection of hepatocytes with a constitutively active form of AMPK. Prolonged AMPK activation triggered apoptosis and activated c-Jun N-terminal kinase (JNK) and caspase-3. Experiments with iodotubercidin, dicoumarol and z-VAD-fmk, which inhibited AMPK, JNK and caspase activation, respectively, supported the notion that prolonged AMPK activation in liver cells induces apoptosis through an activation pathway that involves JNK and caspase-3.

Maturation of the mammalian nervous system requires adequate provision of thyroid hormone and mechanisms that enhance tissue responses to the hormone. Here, we report that the development of cones, the photoreceptors for daylight and color vision, requires protection from thyroid hormone by type 3 deiodinase, a thyroid hormone-inactivating enzyme. Type 3 deiodinase, encoded by Dio3, is expressed in the immature mouse retina. In Dio3(-/-) mice, approximately 80% of cones are lost through neonatal cell death. Cones that express opsin photopigments for response to both short (S) and medium-long (M) wavelength light are lost. Rod photoreceptors, which mediate dim light vision, remain essentially intact. Excessive thyroid hormone in wild-type pups also eliminates cones. Cone loss is mediated by cone-specific thyroid hormone receptor beta2 (TRbeta2) as deletion of TRbeta2 rescues cones in Dio3(-/-) mice. However, rescued cones respond to short but not longer wavelength light because TRbeta2 under moderate hormonal stimulation normally induces M opsin and controls the patterning of M and S opsins over the retina. The results suggest that type 3 deiodinase limits hormonal exposure of the cone to levels that safeguard both cone survival and the patterning of opsins that is required for cone function.