bluuebunny/crossref_metadata_2025_split huggingface dataset:
Abstract 4535: SUV420H1 enhances the phosphorylation and transcription of ERK1 in cancer cells
Authors: Vougiouklakis Theodore , Saloura Vassiliki , Nakamura Yusuke , Hamamoto Ryuji 
Published: July 2016

Abstract
The oncogenic protein ERK1, a member of the extracellular signal-regulated kinase (ERK) cascade, is a signaling molecule involved in various types of human cancer. Functional regulation by phosphorylation of kinases in the ERK pathway has been extensively studied, however methylation of the ERK1 protein has not been reported to date. Over the past decade, a number of protein methyltransferases have been identified and reported to methylate histone proteins as well as non-histone substrates related to various molecular functions that have pivotal roles in tumorigenesis. To identify candidate enzymes that may methylate ERK1, we performed an in vitro methyltransferase assay using various recombinant methyltransferases and found that SUV420H1 methylates ERK1. Mass spectrometry analysis validated our findings and identified ERK1 to be tri-methylated by SUV420H1 at lysines 302 and 361. Given that some protein methylation is known to affect protein phosphorylation, we then investigated the biological importance of the individual K302 and K361 methylated lysine residues on the phosphorylation status of ERK1. Substitution of methylation sites (lysine to arginine) diminished phosphorylation levels of ERK1 (Thr202 and Tyr204), supporting that methylation of these respective residues enhances ERK1 phosphorylation levels. Concordantly, siRNA-mediated SUV420H1 knockdown reduced phosphorylated ERK1 protein levels as visualized on immunoblot, while it interestingly also suppressed ERK1 at the transcript level. SUV420H1 knockdown caused a significant growth-suppressive effect in two squamous cell carcinoma head and neck (SCCHN) cell lines, and flow cytometric cell-cycle analysis showed a significant reduction in the proportion of cells at the S phase, and an increase of those at the G0/G1. Our results indicate that overexpression of SUV420H1 may result in activation of the ERK signaling pathway through enhancement of ERK phosphorylation and transcription, thereby providing new insights in the regulation of the ERK cascade in human cancer.
Citation Format: Theodore Vougiouklakis, Vassiliki Saloura, Yusuke Nakamura, Ryuji Hamamoto. SUV420H1 enhances the phosphorylation and transcription of ERK1 in cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4535.
Abstract 4542: Ube2d family members, Ube2e family members and Ube2w modulate the ubiquitination and degradation of EGFR by Cbl
Authors: Ma Ke , Ryan Philip , Klevit Rachel , Lipkowitz Stanley 
Published: July 2016

Abstract
Ubiquitin ligases (E3s) are critical component of ubiquitination. In collaboration with ubiquitin-conjugating enzymes (E2s), E3s confer specificity to the ubiquitination process and direct the conjugation of ubiquitin to one or more lysines on the target proteins. Cbl proteins are RING finger E3s that play a significant role in regulating activity of many tyrosine kinases (e.g., EGFR, MET) by ubiquitination. In our study, we used enzymatic and yeast two-hybrid assays to characterize the E2s that can interact with Cbl proteins. Using an in vitro E3 assay, we found that only the Ube2d family of E2s mediates autoubiquitination of the Cbl proteins. Subsequently, using the yeast two-hybrid system, we found that the three Ube2e family members and Ube2w interact with the RF domain of Cbl. This suggests that Ube2e and Ube2w are relevant to the ubiquitination and degradation of substrates by Cbl. Knockdown of Ube2w decreases ubiquitination of EGFR in Hela cells. In the in vitro E3 assay we found that Ube2w can monoubiquitinate Cbl and increase autoubiquitination of Cbl mediated by ube2d2. Surprisingly, we found that knockdown of Ube2e increases downregulation and ubiquitination of EGFR in HeLa cells. Mechanistically we found that three Ube2e members inhibit autoubiquitination of Cbl mediated by Ube2d2 in vitro. Further, we showed that Ube2e does not affect ubiquitin charging of Ube2d2 by the ubiquitin-activating enzyme (E1) in vitro. This suggests that Ube2e does not compete with Ube2d2 for the E1 under these conditions. Thus, our data suggest that Ube2e acts as a positive modulator of EGFR signaling by competing for Cbl with Ube2d2 and thus prevents ubiquitination and downregulation of the EGFR by Cbl in combination with Ube2d2. Together these data demonstrate that there is an E2 network which modulates the ubiquitination and downregulation of the EGFR by Cbl.
Citation Format: Ke Ma, Philip Ryan, Rachel Klevit, Stanley Lipkowitz. Ube2d family members, Ube2e family members and Ube2w modulate the ubiquitination and degradation of EGFR by Cbl. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4542.
Abstract 4549: Proteomic analysis of ubiquitination identifies the interplay between HSP90 inhibition and CUL5 in the control of autophagy
Authors: Batista Silvia A a , Samant Rahul , Clarke Paul A. , Workman Paul 
Published: July 2016

Abstract
HSP90 has emerged as an important target in cancer therapy as inhibition of HSP90 can result in the degradation of many oncogenic client proteins. The two major cellular protein degradation pathways are the ubiquitin-proteasome system and autophagy. The role of the ubiquitin-proteasome pathway in client protein degradation following HSP90 inhibition is well established. We recently described how this process is controlled by an E3-ubiquitin ligase, cullin 5 (CUL5). We sought to further understand how both major protein degradation pathways are regulated in response to HSP90 inhibition. To do this, we performed a proteomic screen on the ubiquitination status of proteins extracted from HT29 cells following HSP90 inhibition by 17-AAG and / or siRNA silencing of CUL5 expression. We found the level of ubiquitination of 125 proteins increased by more than 2 fold in response to 17-AAG treatment. For 66 of these proteins, CUL5 silencing eliminated the 17-AAG-induced increase in ubiquitination; for 30 proteins CUL5 silencing partially reduced the 17-AAG-induced increase in ubiquitination; and for 29 proteins CUL5 silencing had no effect. Importantly, we found some known regulators of autophagy to be among those proteins ubiquitinated in response to 17-AAG treatment and rescued by CUL5 silencing. There was a correlation between levels of ubiquitination and levels of protein degradation. We measured markers of autophagic flux, LC3B and p62, and found clear evidence for altered pathway flux in response to HSP90 inhibition and CUL5 silencing. This study indicates that the interplay between HSP90 inhibition and CUL5 can control both proteasomal degradation and autophagy.
Citation Format: Silvia A a Batista, Rahul Samant, Paul A. Clarke, Paul Workman. Proteomic analysis of ubiquitination identifies the interplay between HSP90 inhibition and CUL5 in the control of autophagy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4549.
Abstract 4551: Regulation of replicative stress by deacetylation and dephosphorylation
Authors: Göder Anja , Schäfer Claudia , Nikolova Teodora , Schneider Günter , Krämer Oliver H. 
Published: July 2016

Abstract
Over 3 billion base pairs of a mammalian cell have to be replicated with every cell division. Limitations in the supply of nucleotides and DNA lesions slow down replication fork speed and trigger a complex replicative stress response to prevent genomic instability and cancer development. The checkpoint kinases ataxia telangiectasia mutated (ATM), ATM and Rad3-related (ATR), checkpoint kinase-1 (CHK1), and checkpoint kinase-2 (CHK2) are at the heart of this response. These factors catalyze processes that slow down the cell cycle as well as mechanisms that stabilize the replication fork and processes that initiate DNA repair. Such pathways ensure the faithful transmission of DNA without epigenetic alterations and DNA mutations. As expected for such a pivotal mechanism, checkpoint kinase signaling is highly regulated. Posttranslational modifications including phosphorylation and acetylation regulate checkpoint kinases at multiple levels. Recent data show that class I histone deacetylases (HDACs) can affect checkpoint kinase signaling and genomic stability. How these HDACs control checkpoint kinases exactly and if they show specificity for certain checkpoint kinases and DNA repair pathways has not been resolved. Moreover, although it is known that phosphorylation triggers checkpoint kinase activation and that the trimeric phosphatase PP2A attenuates checkpoint kinase phosphorylation it is unknown how this activity of PP2A is modulated. PP2A consists of the subunits A (structural component, PPP2R1A/B), C (catalytic activity, PPP2CA/B), and B (discriminates between substrates to allow specificity of the PP2A holoenzyme). Our data illustrate that class I HDACs are required for checkpoint kinase phosphorylation in human and murine cells. We show that these enzymes suppress the expression of certain PP2A subunits and we reveal that one of the B type subunits specifically targets checkpoint kinases for dephosphorylation by the PP2A holoenzyme. With genetic and biochemical approaches, including RNAi, CRISPR-Cas9, DNA fiber assays, confocal microscopy, and DNA and protein analyses, we demonstrate how these mechanisms maintain S phase arrest and the survival of cells undergoing replicative stress.
Citation Format: Anja Göder, Claudia Schäfer, Teodora Nikolova, Günter Schneider, Oliver H. Krämer. Regulation of replicative stress by deacetylation and dephosphorylation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4551.
Abstract 4559: Sprouty2 differentially regulates signaling and phenotypic responses of glioblastoma cells to DNA damaging agents and receptor kinase inhibitors
Authors: Sosale Nisha G. , Shin Sally , Lazzara Matthew J. 
Published: July 2016

Abstract
Key to improving glioblastoma patient survival is the exploration of the signaling mechanisms that regulate cellular response to approved and investigational therapeutics, including DNA damaging agents and targeted therapeutics such as EGFR and MET kinase inhibitors. Previous work from our group demonstrated that Sprouty2 (SPRY2) surprisingly acted more as a tumor promoter than tumor suppressor in glioblastoma cell lines and tumor xenografts, with SPRY2 knockdown reducing proliferation of human glioblastoma cells and antagonizing the growth of subcutaneous tumor xenografts in mice. We hypothesized here that these effects may result from a perturbation in the cell cycle and that such effects may augment cell cycle shifts induced by DNA damaging agents or kinase inhibitors. Flow cytometry measurements indicated shifts in DNA content distribution in glioblastoma cell lines consistent with cell cycle arrest in response to SPRY2 knockdown alone, and that these shifts were substantially augmented by carboplatin or to a lesser degree by a combination of EGFR and MET inhibitors, but not by temozolomide. Western blotting indicated that SPRY2 knockdown resulted in increased phosphorylation of Ataxia Telangiectasia Mutated (ATM), a kinase involved in the DNA damage response pathway, at a serine residue (1981) that regulates the dissociation of inactivate ATM homodimers into activated monomers. In response to carboplatin, but not the other therapeutics tested, a robust increase was observed in Checkpoint kinase 1 (CHK1) phosphorylation at serine 345, which has been reported to cause cell cycle arrest. This effect was further enhanced by SPRY2 knockdown, which may help explain why the largest cell cycle perturbations were observed in SPRY2-deficient cells treated with carboplatin. Increased phosphorylation of p38 was also observed in response to carboplatin or EGFR and MET inhibitors. This may have contributed to cell cycle arrest, but may also have cooperated with the inhibition of AKT phosphorylation that occurred only in response to EGFR and MET inhibition to drive cell death, an effect that was also augmented by SPRY2 knockdown. Overall, this work has identified a small network of SPRY2-regulated signaling processes that control phenotypic responses of glioblastoma cells to different clinically relevant therapeutics and provides motivation for further study of the role of SPRY2 in glioblastoma.
Citation Format: Nisha G. Sosale, Sally Shin, Matthew J. Lazzara. Sprouty2 differentially regulates signaling and phenotypic responses of glioblastoma cells to DNA damaging agents and receptor kinase inhibitors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4559.
Abstract 4563: Role of ezrin in PKA dependent colorectal cancer cell fate
Authors: Leiphrakpam Premila , Brattain Michael G. 
Published: July 2016

Abstract
Introduction: Metastasis is the main cause of cancer related death in CRC. However, the molecular basis of CRC metastasis is poorly understood. Ezrin is a known cAMP dependent AKAP. We have found ezrin hyperphosphorylation at T567 is critical for CRC cell survival. In this study we evaluated the dependency of PKA activation with ezrin phosphorylation at T567 on its downstream cell survival mechanisms.
Methods: Small molecule ezrin kinase inhibitor NSC668394 was used to test the effect of inhibition of ezrinT567 phosphorylation on PKA activation and its downstream cell survival signaling. A genetic knockdown approach was utilized to further confirm the result. In another approach, growth factor mediated activation of ezrin T567 phosphorylation was performed and ezrin/PKA signaling was then analyzed for its downstream cell survival effects. To further confirm the findings ezrin phospho-mimetic (T567D) and phospho-deficient (T567A) mutants were generated through site directed mutation approaches.
Results: We demonstrated that dephosphorylation of ezrin at T567 activates PKA in a cAMP independent manner and leads to CRC cell death through inhibition of XIAP and survivin. Moreover, the stable knockdown of ezrin showed similar result. Interestingly, ligand mediated ezrin hyperphosphorylation at T567 has the opposite effect in which there is cAMP dependent PKA activation leading to CRC survival through upregulation of XIAP and survivin. This was further confirmed with ezrin T567 phospho-mimetic and phospho-deficient mutants which demonstrate PKA activation by cAMP dependent and independent mechanisms respectively leading to different CRC cell fates. Therefore, ezrin signaling demonstrated two opposing pathways in which both requires activation of PKA and yet result in different CRC cell fates by regulating XIAP and survivin expression.
Conclusions: Two different ezrin signaling dependent pathways were identified which are dependent on the same enzymatic activity but with opposite effects on CRC cell fates. Therefore, ezrin might be a novel target that could be utilized for the development of new therapeutic strategies and further understanding the mechanism of ezrin/cAMP-PKA activation and its role in CRC cell survival could have potential impact for treatment of distant metastasis in CRC.
Citation Format: Premila Leiphrakpam, Michael G. Brattain. Role of ezrin in PKA dependent colorectal cancer cell fate. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4563.
Abstract 4565: Antizyme inhibitor regulates the expression of proteins involved in mitosis and affects prostate cancer cell growth
Authors: Rice James M. , Kusztos Amanda , Zetter Bruce R. 
Published: July 2016

Abstract
Upregulation of polyamine biosynthesis is a requirement for cell growth and proliferation. Increases in polyamine levels are mediated by the enzyme ornithine decarboxylase (ODC), which is inhibited by antizyme (AZ). Rescue of ODC activity is accomplished by expression of antizyme inhibitor (AZI), which prevents binding of AZ to ODC. Increases in AZI gene copy number, mRNA transcripts, protein expression and AZI nuclear localization are all associated with prostate cancer. Furthermore, knockdown of AZI expression prevents prostate cancer cell growth in vitro and in vivo. To determine if AZI regulation affects prostate cancer cell growth via mechanisms that are independent of polyamide levels, we conducted a proteomic analysis of the AZI interactome by co-IP and MS/MS. This resulted in the identification of 29 high-confidence interacting proteins (HCIPs), 10 of which are associated with microtubules or with the mitotic spindle including TPX2 and KIFC1. Constitutive overexpression of AZI in PC3M-LN4 prostate cancer cells leads to a decrease in expression of the microtubule associated protein KIFC1, further supporting an interaction between AZI and KIFC1. Silencing of AZI gene expression using siRNA nanoparticles results in a decrease in prostate cancer cell proliferation and altered expression of proteins involved in mitosis. Taken together, this data suggests that AZI may influence prostate cancer cell proliferation via mechanisms that are independent of polyamine regulation.
Citation Format: James M. Rice, Amanda Kusztos, Bruce R. Zetter. Antizyme inhibitor regulates the expression of proteins involved in mitosis and affects prostate cancer cell growth. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4565.
Abstract 4568: 14-3-3z-mediated oncogenic signaling downstream of HER2 in breast cancer
Authors: Chan Tsz Yin , Banks Courtney , Peterson Bennet , Andersen Joshua 
Published: July 2016

Abstract
The goal of this study is to identify mechanisms by which the oncogenic tyrosine kinase receptor HER2 promotes the growth and metastasis of breast cancer. Previously published work suggests that the phospho-binding protein 14-3-3ζ, which is highly expressed in a variety of breast cancer subtypes, promotes HER2-mediated oncogenic signaling. In this study, we hypothesized that 14-3-3ζ sustains HER2 signaling by binding to and modulating a network of proteins that are phosphorylated downstream of HER2 activation. Using proteomics as a tool to identify 14-3-3ζ interacting proteins in HER2+ breast cancer cells treated with or without Lapatinib (HER2 inhibitor), we identified a subset HER2-dependent 14-3-3ζ interacting proteins. We have focused our current effort on one of these proteins, SH3BP4, which plays a critical role in controlling mTOR activation. We have identified a nutrient-sensitive phosphorylation within a putative 14-3-3ζ binding site on SH3BP4 and we are currently working to understand its mechanism of regulation and impact on mTOR-mediated growth signaling downstream of HER2.
Citation Format: Tsz Yin Chan, Courtney Banks, Bennet Peterson, Joshua Andersen. 14-3-3z-mediated oncogenic signaling downstream of HER2 in breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4568.
Abstract 4580: A blueprint of androgen receptor splice variant transactivation
Authors: Cao Subing , Xu Duo , Qi Yanfeng , Zhan Yang , Sartor Oliver , Dong Yan 
Published: July 2016

Abstract
Constitutively active androgen receptor splice variants (AR-Vs) have been implicated as one driver of castration-resistant progression of prostate cancer. However, the critical steps leading to AR-V transactivation remain largely unknown. AR-V7 and ARv567es are two major AR-Vs expressed in human prostate cancer specimens. We found that disruption of AR-V7 and ARv567es homodimerization did not affect their nuclear localization, indicating that they can enter the nucleus as monomers. On the other hand, disrupting the heterodimerization between AR-V7 or ARv567es and AR-FL abolished AR-V7 and ARv567es-induced nuclear translocation of AR-FL, indicating that the heterodimerization is required for AR-FL to be piggy bagged into the nucleus. AR-V7 chromatin immunoprecipitation assay further showed that mutating the dimerization interface of AR-V7 did not decrease the ability of AR-V7 to bind to the promoter of its target genes, UBE2C and CCNA2, indicating that dimerization is not required for AR-Vs to bind to DNA. Moreover, mutating the DNA-binding interface of AR-V7 only partially inhibited AR-V7 homodimerization and had no effect on AR-FL/AR-V7 dimerization, suggesting that DNA binding may not be necessary for AR-V/AR-V or AR-FL/AR-V dimerization. Nonetheless, both the dimerization and the DNA-binding mutants of AR-V7 lost trans-activating activity. Taken together, our data suggests that AR-V DNA binding and dimerization are two independent but indispensable steps for AR-V transactivation. AR-V can enter the nucleus as a monomer, and then either forms a dimer before binding to DNA or binds to DNA as a monomer and then forms a dimer to trans-activate gene transcription. Our study may provide insights into developing more precisely targeted strategies to overcome AR-V-driven castration resistance.
Citation Format: Subing Cao, Duo Xu, Yanfeng Qi, Yang Zhan, Oliver Sartor, Yan Dong. A blueprint of androgen receptor splice variant transactivation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4580.