Nuclear Factor of Activated T Cells Balances Angiogenesis Activation and Inhibition

It has been demonstrated that vascular endothelial cell growth factor (VEGF) induction of angiogenesis requires activation of the nuclear factor of activated T cells (NFAT). We show that NFATc2 is also activated by basic fibroblast growth factor and blocked by the inhibitor of angiogenesis pigment epithelial–derived factor (PEDF). This suggests a pivotal role for this transcription factor as a convergence point between stimulatory and inhibitory signals in the regulation of angiogenesis

Nitric Oxide Induces Cell Death by Regulating Anti-Apoptotic BCL-2 Family Members

Nitric oxide (NO) activates the intrinsic apoptotic pathway to induce cell death. However, the mechanism by which this pathway is activated in cells exposed to NO is not known. Here we report that BAX and BAK are activated by NO and that cytochrome c is released from the mitochondria. Cells deficient in Bax and Bak or Caspase-9 are completely protected from NO-induced cell death. The individual loss of the BH3-only proteins, Bim, Bid, Puma, Bad or Noxa, or Bid knockdown in Bim−/−/Puma−/− MEFs, does not prevent NO-induced cell death. Our data show that the anti-apoptotic protein MCL-1 undergoes ASK1-JNK1 mediated degradation upon exposure to NO, and that cells deficient in either Ask1 or Jnk1 are protected against NO-induced cell death. NO can inhibit the mitochondrial electron transport chain resulting in an increase in superoxide generation and peroxynitrite formation. However, scavengers of ROS or peroxynitrite do not prevent NO-induced cell death. Collectively, these data indicate that NO degrades MCL-1 through the ASK1-JNK1 axis to induce BAX/BAK-dependent cell death

Twist1 Suppresses Senescence Programs and Thereby Accelerates and Maintains Mutant Kras-Induced Lung Tumorigenesis

KRAS mutant lung cancers are generally refractory to chemotherapy as well targeted agents. To date, the identification of drugs to therapeutically inhibit K-RAS have been unsuccessful, suggesting that other approaches are required. We demonstrate in both a novel transgenic mutant Kras lung cancer mouse model and in human lung tumors that the inhibition of Twist1 restores a senescence program inducing the loss of a neoplastic phenotype. The Twist1 gene encodes for a transcription factor that is essential during embryogenesis. Twist1 has been suggested to play an important role during tumor progression. However, there is no in vivo evidence that Twist1 plays a role in autochthonous tumorigenesis. Through two novel transgenic mouse models, we show that Twist1 cooperates with KrasG12D to markedly accelerate lung tumorigenesis by abrogating cellular senescence programs and promoting the progression from benign adenomas to adenocarcinomas. Moreover, the suppression of Twist1 to physiological levels is sufficient to cause Kras mutant lung tumors to undergo senescence and lose their neoplastic features. Finally, we analyzed more than 500 human tumors to demonstrate that TWIST1 is frequently overexpressed in primary human lung tumors. The suppression of TWIST1 in human lung cancer cells also induced cellular senescence. Hence, TWIST1 is a critical regulator of cellular senescence programs, and the suppression of TWIST1 in human tumors may be an effective example of pro-senescence therapy

<i>Ask1</i> is required for NO-induced cell death.

<p>Phospho-JNK was measured to assess JNK activity in <i>Ask1^−/−</i> and <i>Bax^−/−/Bak^−/−</i> MEFs treated with 0 or 400 µM DETA-NO (A). Percent LDH release was measured in wild type and Ask<i>1^−/−</i> MEFs treated with 0, 100, 200 and 400 µM DETA-NO (B).</p

The individual loss of the BH3-only proteins BID, BIM, PUMA, BAD and NOXA does not protect against nitric oxide-induced cell death.

<p>Wild type, <i>Bid^−/−</i> (A), <i>Bim^−/−</i> (B), <i>Puma^−/−</i> (C), <i>Bad^−/−</i> (D) and <i>Noxa^−/−</i> (E) MEFs were treated with 0 and 400 µM DETA-NO for 24 and 48 hours. Percent cell death was measured by LDH release.</p

MCL-1 is degraded in response to nitric oxide treatment and is dependent on the ASK1/JNK1 pathway.

<p>MCL-1 protein expression was measured in <i>Bax^−/−/Bak^−/−</i> MEFs treated with 400 µM DETA-NO for 0, 8, 16 and 24 hours (A). MCL-1 degradation was measured in <i>Bax^−/−/Bak^−/−</i> MEFs pre-treated with or without 20 µM MG132 followed by 0 or 400 µM DETA-NO for 24 hours (B). MCL-1 protein expression was measured in <i>Jnk1^−/−</i> (C), <i>Ask1^−/−</i> (D) MEFs treated with 400 µM DETA-NO for the indicated time points.</p

NO-induced apoptosis is not due to ROS or peroxynitrite generation.

<p><i>Bax^−/−/Bak^−/−</i> MEFs adapted to glucose or galactose were treated with 400 µM DETA-NO or 10 µM rotenone for 48 hours and cell death was measured by percent LDH release (A). Wild type MEFs were pre-treated with the ROS inhibitor EUK-134 (20 µM) followed by 0, 100, 200 and 400 µM DETA-NO for 24 hours and cell death was measured by LDH release (B). PEITC is known to generate endogenous ROS and was used as a positive control. Wild type MEFs were pre-treated with the peroxynitrite scavengers, uric acid (1 mM) and ebselen (10 µM) followed by 0, 100, 200 and 400 µM DETA-NO for 24 hours and cell death was measured by percent LDH release (C and D). Wild type MEFs were pre-treated with the nitric oxide scavenger PTIO (1 mM) followed by 0, 100, 200 and 400 µM DETA-NO for 24 hours and cell death was measured by percent LDH release (E).</p

BAX and BAK mediate nitric oxide-indeced cell death.

<p><i>Bax^−/−/Bak^−/−</i> MEFs were infected with either Bax, Bak or GFP as a control. BAX and BAK expression was verified by Western analysis (A,B). <i>Bax^−/−/Bak^−/−</i> MEFs expressing GFP, BAK or BAX were treated with 0, 100, 200 and 400 µM DETA-NO for 48 hours and cell death was measured by LDH release (C).</p