Yingtao Zhang

Deacetylation of cortactin by SIRT1 promotes cell migration.

Cortactin binds F-actin and promotes cell migration. We showed earlier that cortactin is acetylated. Here, we identify SIRT1 (a class III histone deacetylase) as a cortactin deacetylase and p300 as a cortactin acetylase. We show that SIRT1 deacetylates cortactin in vivo and in vitro and that the SIRT1 inhibitor EX-527 increases amounts of acetylated cortactin in ovarian cancer cells. We also show that p300 acetylates cortactin in vivo and that cells lacking or depleted of p300 express less-acetylated cortactin than do control cells. Deletion analysis mapped the SIRT1-binding domain of cortactin to its repeat region, which also binds F-actin. Mouse embryo fibroblasts (MEFs) lacking sir2α (the mouse homolog of SIRT1) migrated more slowly than did wild-type cells. The expression of SIRT1 in sir2α-null cells restored migratory capacity, as did expression of a deacetylation-mimetic mutant of cortactin. SIRT1 and cortactin were more abundant in breast tumor tissue than in their normal counterparts, whereas SIRT1 expression inversely correlates with the ratio of acetylation cortactin versus total cortactin. These data suggest that deacetylation of cortactin is associated with high levels of SIRT1 and tumorigenesis. Finally, breast and ovarian cancer cell lines expressing an acetylation mimetic mutant of cortactin are less motile than that of control cells, whereas cells expressing the deacetylation mimetic mutant of cortactin migrate faster than that of control cells in Transwell migration assays. In summary, our results suggest that cortactin is a novel substrate for SIRT1 and p300 and, for the first time, a possible role for SIRT1 in cell motility through deacetylation of cortactin.

MDM2 Acts Downstream of p53 as an E3 Ligase to Promote FOXO Ubiquitination and Degradation

Members of the FOXO (forkhead O) class of transcription factors are tumor suppressors that also control aging and organismal life span. Mammalian FOXO degradation is proteasome-mediated, although the ubiquitin E3 ligase for FOXO factors remains to be defined. We show that MDM2 binds to FOXO1 and FOXO3A and promotes their ubiquitination and degradation, a process apparently dependent on FOXO phosphorylation at AKT sites and the E3 ligase activity of MDM2. Binding of MDM2 to FOXO occurs through the region of MDM2 that directs its cellular trafficking and the forkhead box of FOXO1. MDM2 promotes the ubiquitination of FOXO1 in a cell-free system, and its knockdown by small interfering RNA causes accumulation of endogenous FOXO3A protein in cells and enhances the expression of FOXO target genes. In cells stably expressing a temperature-sensitive p53 mutant, activation of p53 by shifting to permissive temperatures leads to MDM2 induction and degradation of endogenous FOXO3A. These data suggest that MDM2 acts as an ubiquitin E3 ligase, downstream of p53, to regulate the degradation of mammalian FOXO factors.

A kinome screen identifies checkpoint kinase 1 (CHK1) as a sensitizer for RRM1-dependent gemcitabine efficacy.

Gemcitabine is among the most efficacious and widely used antimetabolite agents. Its molecular targets are ribonucleotide reductase M1 (RRM1) and elongating DNA. Acquired and de novo resistance as a result of RRM1 overexpression are major obstacles to therapeutic efficacy. We deployed a synthetic lethality screen to investigate if knockdown of 87 selected protein kinases by siRNA could overcome RRM1-dependent gemcitabine resistance in high and low RRM1-expressing model systems. The models included genetically RRM1-modified lung and breast cancer cell lines, cell lines with gemcitabine-induced RRM1 overexpression, and a series of naturally gemcitabine-resistant cell lines. Lead molecular targets were validated by determination of differential gemcitabine activity using cell lines with and without target knock down, and by assessing synergistic activity between gemcitabine and an inhibitor of the lead target. CHK1 was identified has the kinase with the most significant and robust interaction, and it was validated using AZD7762, a small-molecule ATP-competitive inhibitor of CHK1 activation. Synergism between CHK1 inhibition and RRM1-dependent gemcitabine efficacy was observed in cells with high RRM1 levels, while antagonism was observed in cells with low RRM1 levels. In addition, four cell lines with natural gemcitabine resistance demonstrated improved gemcitabine efficacy after CHK1 inhibition. In tumor specimens from 187 patients with non-small-cell lung cancer, total CHK1 and RRM1 in situ protein levels were significantly (p = 0.003) and inversely correlated. We conclude that inhibition of CHK1 may have its greatest clinical utility in malignancies where gemcitabine resistance is a result of elevated RRM1 levels. We also conclude that CHK1 inhibition in tumors with low RRM1 levels may be detrimental to gemcitabine efficacy.

Rab1 in cell signaling, cancer and other diseases

The endoplasmic reticulum (ER) and Golgi membrane system have major roles in cell signaling and regulation of the biosynthesis/transport of proteins and lipids in response to environmental cues such as amino acid and cholesterol levels. Rab1 is the founding member of the Rab small GTPase family, which is known to mediate dynamic membrane trafficking between ER and Golgi. Growing evidence indicate that Rab1 proteins have important functions beyond their classical vesicular transport functions, including nutrient sensing and signaling, cell migration and presentation of cell-surface receptors. Moreover, deregulation of RAB1 expression has been linked to a myriad of human diseases such as cancer, cardiomyopathy and Parkinson’s disease. Further investigating these new physiological and pathological functions of Rab1 should provide new opportunities for better understanding of the disease processes and may lead to more effective therapeutic interventions.

Quantitative analysis of intracellular nucleoside triphosphates and other polar metabolites using ion pair reversed-phase liquid chromatography coupled with tandem mass spectrometry

Simultaneous, quantitative determination of intracellular nucleoside triphosphates and other polar metabolites using liquid chromatography with electrospray ionization tandem mass spectrometry (LC-MS/MS) represents a bioanalytic challenge because of charged, highly hydrophilic analytes presented at a large concentration range in a complex matrix. In this study, an ion pair LC-MS/MS method using triethylamine (TEA)-hexafluoroisopropanol (HFIP) ion-pair mobile phase was optimized and validated for simultaneous and unambiguous determination of 8 nucleoside triphosphates (including ATP, CTP, GTP, UTP, dATP, dCTP, dGTP, and dTTP) in cellular samples. Compared to the the less volatile ion-pair reagent, triethylammonium acetate (100mM, pH 7.0), the combination of HFIP (100mM) and TEA (8.6mM) increased the MS signal intensity by about 50-fold, while retaining comparable chromatographic resolution. The isotope-labeled internal standard method was used for the quantitation. Lower limits of quantitation were determined at 0.5nM for CTP, UTP, dATP, dCTP, and dTTP, at 1nM for ATP, and at 5nM for GTP and dGTP. The intra- and inter-day precision and accuracy were within the generally accepted criteria for bioanalytical method validation (<15%). While the present method was validated for the quantitation of intracellular nucleoside triphosphates, it had a broad application potential for quantitative profiling of nucleoside mono- and bi-phosphates as well as other polar, ionic metabolic intermediates (including carbohydrate derivatives, carboxylic acid derivatives, co-acyl A derivatives, fatty acyls, and others) in biological samples.

Ubiquitination and Degradation of Ribonucleotide Reductase M1 by the Polycomb Group Proteins RNF2 and Bmi1 and Cellular Response to Gemcitabine

Ribonucleotide reductase M1 (RRM1) is required for mammalian deoxyribonucleotide (dNTP) metabolism. It is the primary target of the antimetabolite drug gemcitabine, which is among the most efficacious and most widely used cancer therapeutics. Gemcitabine directly binds to RRM1 and irreversibly inactivates ribonucleotide reductase. Intra-tumoral RRM1 levels are predictive of gemcitabine’s therapeutic efficacy. The mechanisms that regulate intracellular RRM1 levels are largely unknown. Here, we identified the E3 ubiquitin-protein ligases RNF2 and Bmi1 to associate with RRM1 with subsequent poly-ubiquitination at either position 48 or 63 of ubiquitin. The lysine residues 224 and 548 of RRM1 were identified as major ubiquitination sites. We show that ubiquitinated RRM1 undergoes proteasome-mediated degradation and that targeted post-transcriptional silencing of RNF2 and Bmi1 results in increased RRM1 levels and resistance to gemcitabine. Immunohistochemical analyses of 187 early-stage lung cancer tumor specimens revealed a statistically significant co-expression of RRM1 and Bmi1. We were unable to identify suitable reagents for in situ quantification of RNF2. Our findings suggest that Bmi1 and possibly RNF2 may be attractive biomarkers of gemcitabine resistance in the context of RRM1 expression. They also provide novel information for the rational design of gemcitabine-proteasome inhibitor combination therapies, which so far have been unsuccessful if given to patients without taking the molecular context into account.

Discoidin Domain Receptors in Lung Cancer

Lung cancer is the leading cause of cancer deaths in the United States and Europe. Tyrosine kinases, involved in signal transduction pathways, are frequently mutated in lung cancer, and novel therapeutic interventions have substantially improved the outcome for patients whose tumors have detectable mutations. The discoidin domain receptors (DDRs) are members of the receptor tyrosine kinase (RTK) family. They are activated by collagen, and mounting evidence from biological model systems suggests key roles for these receptors in tumor cell invasion, migration, and metastatic spread. Several investigations have described associations between expression levels or mutations of DDRs and patients’ survival and response to therapy. However, results from these studies are inconsistent and sometimes contradictory, which may be explained by differences in the use of analytic methods, the retrospective nature of study reports, and differences in biospecimen procurement, storage, and processing.

Abstract 5537: Ubiquitination of RRM1 by Ring1B (RNF2) promotes its degradation and nuclear export

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL RRM1 (ribonucleotide reductase M1) is a key enzyme involved in deoxyribonucleotide (dNTP) synthesis and DNA damage repair. It also plays a critical role in acquired resistance to the chemotherapy drug gemcitabine, which directly inhibits the RR holoenzyme. Despite its significant importance, however, the mechanisms that control RRM1 abundance and subcellular localization are unclear. We have identified a ubiquitin E3 ligase, Ring1B (RNF2), as an RRM1-associated protein in a yeast two-hybrid screen. RRM1 interacts with Ring1B both in endogenous and exogenous situations. Additionally, immunofluorescent staining has shown that Ring1B clearly co-localized with RRM1 in a pattern of diffuse nuclear speckles. We found that the protein level of RRM1 is regulated by a proteasome-mediated degradation pathway. Ring1B induces poly-ubiquitination of RRM1 in vivo. Formation of poly-ubiquitin chains on RRM1 is through both Lys48 (K48) and Lys63 (K63) of the ubiquitin. Our study demonstrates that the cytoplasmic fraction of RRM1 was increased upon Ring1B or ubiquitin overexpression, but significantly decreased after nuclear export inhibition by leptomycin B (LMB). This indicates that RRM1 ubiquitination promotes the translocation of RRM1 from the nucleus to the cytoplasm. We also show that hydrogen peroxide (H2O2) promotes the degradation of RRM1 in dose and time-dependent manners. H2O2 can increase the poly-ubiquitination of RRM1, which suggests that RRM1 ubiquitination may participate in cellular responses to oxidative stress-induced DNA damage repair. In summary, our data propose that Ring1B-induced ubiquitination regulates RRM1 activity through RRM1 degradation and nuclear export. 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 5537. doi:10.1158/1538-7445.AM2011-5537