Elizabeth Johnson

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.

Phytochrome is a family of photoreceptors that regulates plant photomorphogenesis; the best-characterized member of this family is phytochrome A. Here, we report the identification of novel mutations at three Arabidopsis loci (fhy1, fhy2, and fhy3) that confer an elongated hypocotyl in far-red but not in white light. fhy2 mutants are phytochrome A deficient, have reduced or undetectable levels of PHYA transcripts, and contain structural alterations within the PHYA gene. When grown in white light, fhy2 mutants are morphologically indistinguishable from wild-type plants. Thus, phytochrome A appears to be dispensable in white light-grown Arabidopsis plants. fhy2 alleles confer partially dominant phenotypes in far-red light, suggesting that the relative abundance of phytochrome A can affect the extent of the far-red-mediated hypocotyl growth inhibition response. Plants homozygous for the recessive fhy1 and fhy3 mutations have normal levels of functional phytochrome A. The FHY1 and FHY3 gene products may be responsible for the transduction of the far-red light signal from phytochrome A to downstream processes involved in hypocotyl growth regulation.

We show, for the first time, that the tumor suppressor PTEN can have tumor-promoting properties. We show that PTEN acquires these unexpected properties by enhancing gain-of-function mutant p53 (mut-p53) protein levels. We find that PTEN restoration to cells harboring mut-p53 leads to induction of G(1)-S cell cycle progression and cell proliferation and to inhibition of cell death. Conversely, PTEN inhibition in cells expressing wild-type PTEN and mut-p53 leads to inhibition of cell proliferation and inhibition of in vivo tumor growth. We show the dependency of the tumor-promoting effects of PTEN on mut-p53 by showing that knockdown of mut-p53 expression inhibits or reverses the tumor-promoting effects of PTEN. Mechanistically, we show that PTEN expression enhances mut-p53 protein levels via inhibition of mut-p53 degradation by Mdm2 and possibly also via direct protein binding. These findings describe a novel function of PTEN and have important implications for experimental and therapeutic strategies that aim at manipulating PTEN or p53 in human tumors. They suggest that the mutational status of PTEN and p53 should be considered to achieve favorable therapeutic outcomes. The findings also provide an explanation for the low frequency of simultaneous mutations of PTEN and p53 in human cancer.

The tyrosine kinase receptor c-Met and its ligand hepatocyte growth factor (HGF) are frequently overexpressed and the tumor suppressor PTEN is often mutated in glioblastoma. Because PTEN can interact with c-Met-dependent signaling, we studied the effects of PTEN on c-Met-induced malignancy and associated molecular events and assessed the potential therapeutic value of combining PTEN restoration approaches with HGF/c-Met inhibition. We studied the effects of c-Met activation on cell proliferation, cell cycle progression, cell migration, cell invasion, and associated molecular events in the settings of restored or inhibited PTEN expression in glioblastoma cells. We also assessed the experimental therapeutic effects of combining anti-HGF/c-Met approaches with PTEN restoration or mTOR inhibition. PTEN significantly inhibited HGF-induced proliferation, cell cycle progression, migration, and invasion of glioblastoma cells. PTEN attenuated HGF-induced changes of signal transduction proteins Akt, GSK-3, JNK, and mTOR as well as cell cycle regulatory proteins p27, cyclin E, and E2F-1. Combining PTEN restoration to PTEN-null glioblastoma cells with c-Met and HGF inhibition additively inhibited tumor cell proliferation and cell cycle progression. Similarly, combining a monoclonal anti-HGF antibody (L2G7) with the mTOR inhibitor rapamycin had additive inhibitory effects on glioblastoma cell proliferation. Systemic in vivo delivery of L2G7 and PTEN restoration as well as systemic in vivo deliveries of L2G7 and rapamycin additively inhibited intracranial glioma xenograft growth. These preclinical studies show for the first time that PTEN loss amplifies c-Met-induced glioblastoma malignancy and suggest that combining anti-HGF/c-Met approaches with PTEN restoration or mTOR inhibition is worth testing in a clinical setting.