c 14-3-3 promotes cell survival via dynamic interactions with a vast network of binding partners, many of which are involved in stress regulation. We show here that hypoxia (low glucose and oxygen) triggers a rearrangement of the 14-3-3 interactome to favor an interaction with the core autophagy regulator Atg9A. Our data suggest that the localization of mammalian Atg9A to autophagosomes requires phosphorylation on the C terminus of Atg9A at S761, which creates a 14-3-3 docking site. Under basal conditions, this phosphorylation is maintained at a low level and is dependent on both ULK1 and AMPK. However, upon induction of hypoxic stress, activated AMPK bypasses the requirement for ULK1 and mediates S761 phosphorylation directly, resulting in an increase in 14-3-3 interactions, recruitment of Atg9A to LC3-positive autophagosomes, and enhanced autophagosome production. These data suggest a novel mechanism whereby the level of autophagy induction can be modulated by AMPK/ULK1-mediated phosphorylation of mammalian Atg9A.

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Background: The aim of the study was to investigate any associations between benign prostate hyperplasia (BPH) and single nucleotide polymorphisms (SNPs) in the VDR gene (FokI, BsmI, ApaI and TaqαI loci) and the CYP17 gene (MspA1I locus), as well as TA repeat polymorphism in SRD5A2 gene among Lebanese men. Materials and Methods: DNA extracted from blood of 68 subjects with confirmed BPH and 79 age-matched controls was subjected to PCR/PCR-restriction fragment length polymorphism analysis. The odds ra=tio (OR) of having a genotype and the relative risk (RR) of developing BPH for having the genotype were calculated and the alleles were designated risk-bearing or protective. Results: Our data indicated that the A and B alleles of the VDR ApaI and BsmI SNPs were highly associated with increased risk of BPH (p=0.0168 and 0.0002, respectively). Moreover, 63% of the controls compared to 43% of the subjects with BPH were homozygous for none of the risk-bearing alleles (p=0.0123) whereas 60% of the controls and 28% of the subjects with BPH were homozygous for two or more protective alleles (p<0.0001). Conclusions: For the first time, our study demonstrated that ApaI and BsmI of the VDR gene are associated with risk of BPH among Lebanese men. Our study also indicated that overall polymorphism profile of all the genes involved in prostate physiology could be a better predictor of BPH risk.

In this study, we employed proteomics to identify mechanisms of posttranslational regulation on cell survival signaling proteins. We focused on Cu-Zn superoxide dismutase (SOD1), which protects cells from oxidative stress. We found that acylation of K122 on SOD1, while not impacting SOD1 catalytic activity, suppressed the ability of SOD1 to inhibit mitochondrial metabolism at respiratory complex I. We found that deacylase depletion increased K122 acylation on SOD1, which blocked the suppression of respiration in a K122-dependent manner. In addition, we found that acyl-mimicking mutations at K122 decreased SOD1 accumulation in mitochondria, initially hinting that SOD1 may inhibit respiration directly within the intermembrane space (IMS). However, surprisingly, we found that forcing the K122 acyl mutants into the mitochondria with an IMS-targeting tag did not recover their ability to suppress respiration. Moreover, we found that suppressing or boosting respiration levels toggled SOD1 in or out of the mitochondria, respectively. These findings place SOD1-mediated inhibition of respiration upstream of its mitochondrial localization. Lastly, deletion-rescue experiments show that a respiration-defective mutant of SOD1 is also impaired in its ability to rescue cells from toxicity caused by SOD1 deletion. Together, these data suggest a previously unknown interplay between SOD1 acylation, metabolic regulation, and SOD1-mediated cell survival.

Several lines of evidence suggest that protein lysine acetylation pathways are deregulated in cancer (1). Moreover, deacetylase inhibitors are emerging as important anti-tumor therapeutics, suggesting that the forced reprogramming of protein-lysine acetylation is toxic to tumor cells. In this study we show that Sirt1, an NAD+-dependent Sirtuin deacetylase that promotes cancer cell survival, is aberrantly mislocalized to the cytoplasm of breast tumor cells. Moreover, the depletion of cytosolic Sirt1 by siRNA sensitizes breast tumor cells to paclitaxel-induced death. Previously, we developed a biotin-switch proteomics approach to identify cytosolic Sirt1 substrates (2). This approach yielded a variety of substrates with roles in metabolism, survival, and oxidative stress signaling. Our current work focuses on three of the proteins identified as Sirt1 substrates: SOD1, DJ-1, and 14-3-3z. SOD1 and DJ-1 both suppress oxidative stress-induced death, and high levels of 14-3-3z expression suppress chemotherapy-induced apoptosis and correlate with negative patient outcomes in breast cancer. Our preliminary results suggest that acetylation of DJ-1 and SOD1 suppress their anti-oxidant functions, while acetylation of 14-3-3z disrupts its binding to pro-survival proteins. Taken together, our data support a model in which cytosolic Sirt1 activates multiple pathways that work together to promote tumor cell survival. Citation Format: Jeffrey B. Mortenson, Vajira K. Weerasekara, Josh Andersen. Sirt1-mediated suppression of cell death in breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 850. doi:10.1158/1538-7445.AM2013-850