Rhythmic Control of the ARF-MDM2 Pathway by ATF4 Underlies Circadian Accumulation of p53 in Malignant Cells Michiko Horiguchi1Michiko Horiguchi1,2, Satoru Koyanagi1, Ahmed M. Hamdan1, Keisuke Kakimoto1,2Satoru Koyanagi1Ahmed M. Hamdan1Keisuke Kakimoto1 Naoya Matsunaga1Naoya Matsunaga1, Chikamasa Yamashita2, and Shigehiro Ohdo1Chikamasa Yamashita2Shigehiro Ohdo1 Department of Pharmaceutics, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka The sensitivity of cancer cells to chemotherapeutic agents varies according to circadian time. Most chemotherapeutic agents ultimately cause cell death through cell-intrinsic pathways as an indirect consequence of DNA damage. The p53 tumor suppressor gene (TRP53) configures the cell deaths induced by chemotherapeutic agents. In this study, we show that the transcription factor ATF4, a component of the mammalian circadian clock, functions in circadian accumulation of p53 protein in tumor cells. In murine fibroblast tumor cells, ATF4 induced the circadian expression of p19ARF (Cdkn2a). Oscillation of p19ARF interacted in a time-dependent manner with MDM2, a specific ubiquitin ligase of p53, resulting in a rhythmic prevention of its degradation by MDM2. Consequently, the half-life of p53 protein varied in a circadian time-dependent manner without variation in mRNA levels. The p53 protein accumulated during those times when the p19ARF–MDM2 interaction was facilitated. Notably, the ability of the p53 degradation inhibitor nutlin-3 to kill murine fibroblast tumor cells was enhanced when the drug was administered at those times of day during which p53 had accumulated. Taken together, these results suggested that ATF4-mediated regulation of the p19ARF–MDM2 pathway underlies the circadian accumulation of p53 protein in malignant cells. Furthermore, they suggest an explanation for how the sensitivity of cancer cells to chemotherapeutic agents is enhanced at those times of day when p53 protein has accumulated, as a result of circadian processes controlled by ATF4. Cancer Res; 73(8); 2639–49. ©2013 AACR.

AMPK Activation by Oncogenesis Is Required to Maintain Cancer Cell Proliferation in Astrocytic Tumors Marcos Ríos1Marcos Ríos1, Marc Foretz4, Benoit Viollet4, Angel Prieto3, Máximo Fraga2, Jose A. Costoya1, and Rosa Señarís1Marc Foretz4Benoit Viollet4Angel Prieto3Máximo Fraga2Jose A. Costoya1Rosa Señarís1 5′-AMP-activated protein kinase (AMPK) is an energy sensor that controls cell metabolism, and it has been related to apoptosis and cell-cycle arrest. Although its role in metabolic homeostasis is well documented, its function in cancer is much less clear. In this study, we examined the role of AMPK in a mouse model of astrocytoma driven by oncogenic H-Ras V12 and/or with PTEN deletion based on the common constitutive activation of the Raf/MEK/ERK and PI3K/AKT cascades in human astrocytomas. We also evaluated the activity and role of AMPK in human glioblastoma cells and xenografts. AMPK was constitutively activated in astrocytes expressing oncogenic H-Ras V12 in parallel with high cell division rates. Genetic deletion of AMPK or attenuation of its activity in these cells was sufficient to reduce cell proliferation. The levels of pAMK were always related to the levels of phosphorylated retinoblastoma (Rb) at Ser804, which may indicate an AMPK-mediated phosphorylation of Rb. We confirmed this AMPK–Rb relationship in human glioblastoma cell lines and xenografts. In clinical specimens of human glioblastoma, elevated levels of activated AMPK appeared especially in areas of high proliferation surrounding the blood vessels. Together, our findings indicate that the initiation and progression of astrocytic tumors relies upon AMPK-dependent control of the cell cycle, thereby identifying AMPK as a candidate therapeutic target in this setting. Cancer Res; 73(8); 2628–38. ©2013 AACR.

Bone Morphogenic Protein Signaling Is a Major Determinant of Dentate Development Youngshik Choe1, Anastasiia Kozlova5, Daniel Graf5, and Samuel J. Pleasure1,2,3,4 Abstract To understand life-long neurogenesis in the dentate gyrus (DG), characterizing dentate neural stem cells and the signals controlling their development are crucial. In the present study, we show that bone morphogenic protein (Bmp) signaling is a critical regulator of embryonic dentate development, required for initiating neurogenesis in embryonic DG progenitors and required for the establishment of dentate neural stem cells postnatally. We tested the hypothesis that Bmp signaling regulates dentate development in part by controlling the expression of Lef1, a Wnt responsive transcription factor expressed in dentate stem cells and absolutely required for dentate granule cell production. Bmp activation through the Acvr1 receptor induced Lef1 expression and neurogenesis in the embryonic DG. Ectopic expression of Bmp7 in the embryonic midline increased DG neurogenesis and inhibition of local Bmp signaling decreased embryonic DG neurogenesis. Mice with selective loss of Bmp expression due to defective meningeal development or with selective conditional deletion of meningeal Bmp7 also have dentate developmental defects. Conditional deletion of Activin receptor type I (Acvr1) or Smad4 (a downstream target nuclear effector of Bmp signaling) in DG neural stem cells resulted in defects in the postnatal subgranular zone and reduced neurogenesis. These results suggest that Acvr1-mediated meningeal Bmp signaling regulates Lef1 expression in the dentate, regulating embryonic DG neurogenesis, DG neural stem cell niche formation, and maintenance. KJKJ

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