The non-POU domain-containing octamer-binding protein (NONO, also referred to as p54nrb) is a multifunctional nuclear protein engaging in transcriptional regulation, mRNA splicing, nuclear retention of defective RNA, and DNA repair. Emerging evidence has demonstrated that p54nrb is subjected to various posttranslational modifications, including phosphorylation and methylation, which may be important regulators of its multifunction. However, among these modifications, direct evidence of p54nrb acetylation and its underlying mechanism remains unclear. In this study, we reported that lysine 371 of p54nrb was reversibly acetylated by the acetyltransferase general control non-depressible 5 (GCN5) and deacetylase sirtuin 1 (SIRT1), which was crucial for activity of p54nrb to inhibit interleukin-8 (IL-8) expression. Mechanistically, GCN5-mediated acetylation attenuates the recruitment of p54nrb on its core binding motif within the IL-8 gene promoter, preferentially increasing the expression of the IL-8 gene. In contrast, deacetylation by SIRT1 reverses this process. Altogether, our data suggest that reversible acetylation is an important switch for the multiple nuclear functions of p54nrb/NONO.

; PBMC: peripheral blood mononuclear cells; PBS: phosphate buffered saline; PMN: polymorphonuclear neutrophil; PPARA: peroxisome proliferator activated receptor alpha; ROS: reactive oxygen species; SIRT3: sirtuin 3; TB: tuberculosis; TEM: transmission electron microscopy; TFEB: transcription factor EB; TNF: tumor necrosis factor.

Abstract Purpose of the study: The purpose of the study is to identify new effectors of K-Ras mediated and KSR1 dependent colon tumorigenesis. Background: Gene targeting and RNA interference identified Kinase suppressor of Ras1 (KSR1) as an effector of oncogenic Ras-induced tumor formation in mouse embryonic fibroblast (MEF) and human colon tumor cells but dispensable for normal development. Therefore, KSR1 and its downstream signaling molecules are potential targets for therapy in colon tumorigenesis. Previous analyses identified nuclear co-activator Peroxisome proliferator-activated receptor gamma co-activator 1(PGC1) and transcription factor Estrogen Related Receptor α (ERRα) as effectors of Ras-induced and KSR1-dependent cell transformation in MEFs. To identify molecules that are essential for Ras-driven and KSR1-dependent colon tumorigenesis, we performed high-throughput screens with the human colon tumor cell line HCT116, using genome-wide siRNA and natural product libraries. The siRNA screen identified AMP-activated protein kinase subunit γ1 (AMPKγ1) as an effector of KSR1-dependent signaling. Results: Compared to non-targeting siRNA, RNAi of AMPKγ1 in HCT116 cells decreased anchorage-independent growth in polyHEMA-coated plates by 6-fold in 24 h (p<10-3) and by 11-fold in 48 hours (p<10-3). Moreover, RNAi of AMPKγ1 differentially induced apoptosis of HCT116 cells (21%) compared to immortalized normal human colon epithelial cells (HCEC) (0.7%; p<10-4). Similarly, RNAi of AMPK catalytic α subunits (AMPKα1/AMPKα2) induced the death of HCT116 cells (21.5%) compared to non-targeting control siRNA (1.3%). RNAi of K-Ras, KSR1 and AMPKγ1 decreased protein expression of PGC1β and ERRα in HCT116 cells. Protein and mRNA expression of PGC1β and ERRα were significantly up-regulated in colon cancer cell lines compared to HCEC, indicating these are important effectors of K-Ras mediated KSR1 and AMPK-driven cell transformation. Furthermore, stable knockdown of PGC1β and ERRα decreased colony formation by 7-fold (p<10-3) and 3.5-fold (p<10-3) in soft agar and delayed tumor formation in nude mice (p<10-4). In-situ hybridization in human tumor samples revealed PGC1β mRNA to be significantly up-regulated in primary tumor stage I, stage II, and stage IV metastatic tumor samples (p<0.05) compared to normal colonic epithelium. ERRα mRNA was up-regulated in primary tumor stage I, II, III and stage IV metastatic tumors (p<0.05). Assay of natural product library for molecules that phenocopy RNAi of KSR1 and AMPK inhibitor Compound C identified 4-OH-Staurosporine. 4-OH-Staurosporine is a competitive inhibitor ATP binding to recombinant AMPK (α1β1γ1) (Ki=23.5 nM). 4-OH-Staurosporine treatment decreased phosphorylation of AMPK substrates acetyl-CoA carboxylase (ACC) at Ser89 and Raptor at Ser792 and expression of PGC1β and ERRα in a panel of human colon tumor cell lines (HCT116, HCT15, SW480, CACO2, GEO). Treatment with 3.75μM 4-OH-Staurosporine also decreased viability of colon tumor cells (HCT116, 52%; HCT15, 15%; SW480, 12%, CACO2, 58%; DLD1, 16%; GEO, 15%) compared with HCEC (p<10-3). Conclusion: These results demonstrate the importance of AMPK and its downstream signaling effectors PGC1β and ERRα in maintaining colon tumor cell survival driven by oncogenic Ras and demonstrate the value of using KSR1 as a reference standard to detect genes essential to colon tumor survival. Citation Format: Binita Das, Kurt Fisher, Hyunseok Kim, Deanna J. Volle, Deandra R. Smith, Robert S. Livergood, John MacMillan, Michael White, Robert Lewis. Novel effectors of K-Ras-mediated and KSR1 dependent colon tumorigenesis. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr A07. doi: 10.1158/1557-3125.RASONC14-A07

Abstract In budding yeast, Sir2 is a NAD+-dependent histone deacetylase that plays a role in chromatin silencing, longevity and genomic stability. In mammals, sirtuins exist as a family of 7 members (SIRT1-7) that deacetylate histones and many nonhistone proteins. Currently, all sirtuins have been knocked out by gene targeting, and the mutant mice suffer various abnormalities in cell growth, apoptosis, neurological behavior, adaptation to calorie restriction, organ metabolism and function, and/or premature aging, highlighting critical roles of sirtuins in these biological processes. Of note, mice carrying mutations of SIRT1, SIRT2 or SIRT3 also developed cancers, predominantly in the mammary glands and the liver that are accompanied by genomic instability and/or abnormal energy metabolism. Our further analysis revealed distinct role of these siruins in maintaining genome integrity. SIRT1 mutant cells displayed altered histone modification, impaired DNA damage response, and reduced ability to repair DNA damage [1]; SIRT3 mutant cells had increased stress-induced superoxide levels and genomic instability [2]; and SIRT2 loss primarily affects mitotic functions. We found that SIRT2 plays a role in maintaining normal mitosis progression through modulating expression levels of several mitotic regulators, including Aurora-A and -B. Loss of SIRT2, consequently, causes centrosome amplification, aneuploidy, and mitotic catastrophe. SIRT2-deficient mice initially develop normally, however, suffer mammary tumors and hepatocellular carcinoma (HCC) when they become old. These data highlight how accurate maintenance of genomic integrity is critical for the suppression of tumorigenesis. Finally, we found that human breast cancer and HCC exhibit reduced levels of SIRT1-3. These results implicate SIRT1-3 as tumor suppressors in these organs. References: 1. Wang RH, Sengupta K, Li C. et al. Impaired DNA damage response, genome instability, and tumorigenesis in SIRT1 mutant mice. Cancer Cell. 2008; 14: 312-323. 2. Kim HS, Patel K, Muldoon-Jacobs K. et al. SIRT3 is a mitochondria-localized tumor suppressor required for maintenance of mitochondrial integrity and metabolism during stress. Cancer Cell. 2010; 17: 41-52. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr SY11-01. doi:10.1158/1538-7445.AM2011-SY11-01

Insulin resistance is a major risk factor for type 2 diabetes mellitus. The protein encoded by the sirtuin 1 (Sirt1) gene, which is a mouse homolog of yeast Sir2, is implicated in the regulation of glucose metabolism and insulin sensitivity; however, the underlying mechanism remains elusive. Here, using mice with a liver-specific null mutation of Sirt1, we have identified a signaling pathway involving Sirt1, Rictor (a component of mTOR complex 2 [mTorc2]), Akt, and Foxo1 that regulates gluconeogenesis. We found that Sirt1 positively regulates transcription of the gene encoding Rictor, triggering a cascade of phosphorylation of Akt at S473 and Foxo1 at S253 and resulting in decreased transcription of the gluconeogenic genes glucose-6-phosphatase (G6pase) and phosphoenolpyruvate carboxykinase (Pepck). Liver-specific Sirt1 deficiency caused hepatic glucose overproduction, chronic hyperglycemia, and increased ROS production. This oxidative stress disrupted mTorc2 and impaired mTorc2/Akt signaling in other insulin-sensitive organs, leading to insulin resistance that could be largely reversed with antioxidant treatment. These data delineate a pathway through which Sirt1 maintains insulin sensitivity and suggest that treatment with antioxidants might provide protection against progressive insulin resistance in older human populations.

Glucose homeostasis in mammals is mainly regulated by insulin signaling. It was previously shown that SIRT6 mutant mice die before 4 weeks of age, displaying profound abnormalities, including low insulin, hypoglycemia, and premature aging. To investigate mechanisms underlying the pleiotropic phenotypes associated with SIRT6 deficiency, we generated mice carrying targeted disruption of SIRT6. We found that 60% of SIRT6(-/-) animals had very low levels of blood glucose and died shortly after weaning. The remaining animals, which have relatively higher concentrations of glucose, survived the early post-weaning lethality, but most died within one year of age. Significantly, feeding the mice with glucose-containing water increased blood glucose and rescued 83% of mutant mice, suggesting that the hypoglycemia is a major cause for the lethality. We showed that SIRT6 deficiency results in more abundant membrane association of glucose transporters 1 and 4, which enhances glucose uptake. We further demonstrated that SIRT6 negatively regulates AKT phosphorylation at Ser-473 and Thr-308 through inhibition of multiple upstream molecules, including insulin receptor, IRS1, and IRS2. The absence of SIRT6, consequently, enhances insulin signaling and activation of AKT, leading to hypoglycemia. These data uncover an essential role of SIRT6 in modulating glucose metabolism through mediating insulin sensitivity.

Cellular longevity is a complex process relevant to age-related diseases including but not limited to chronic illness such as diabetes and metabolic syndromes. Two gene families have been shown to play a role in the genetic regulation of longevity; the Sirtuin and FOXO families. It is also established that nuclear Sirtuins interact with and under specific cellular conditions regulate the activity of FOXO gene family proteins. Thus, we hypothesize that a mitochondrial Sirtuin (SIRT3) might also interact with and regulate the activity of the FOXO proteins. To address this we used HCT116 cells overexpressing either wild-type or a catalytically inactive dominant negative SIRT3. For the first time we establish that FOXO3a is also a mitochondrial protein and forms a physical interaction with SIRT3 in mitochondria. Overexpression of a wild-type SIRT3 gene increase FOXO3a DNA-binding activity as well as FOXO3a dependent gene expression. Biochemical analysis of HCT116 cells over expressing the deacetylation mutant, as compared to wild-type SIRT3 gene, demonstrated an overall oxidized intracellular environment, as monitored by increase in intracellular superoxide and oxidized glutathione levels. As such, we propose that SIRT3 and FOXO3a comprise a potential mitochondrial signaling cascade response pathway.