bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4468: Targeted deep sequencing of plasma circulating cell-free DNA shows VIM and FBLN1 methylation as potential biomarkers for hepatocellular carcinoma
Authors: Holmila Reetta , Sklias Athena , Muller David C. , Degli Esposti Davide , Guilloreau Paule , Mckay James , Sangrajrang Suleeporn , Srivatanakul Petcharin , Hainaut Pierre , Merle Philippe , Nogueira da Costa Andre , Herceg Zdenko
Published: July 2016

Hepatocellular carcinoma (HCC) is the second most common cause of cancer death worldwide, but is still lacking sensitive and specific biomarkers for diagnosis and prognosis. HCC carcinogenesis is a multi-step process that usually arises in background chronic metabolic, inflammatory and/or infectious liver disease and it exhibits a wide range of epigenetic alterations that could potentially be used as biomarkers. In this study, we applied the targeted massively parallel semiconductor sequencing to assess in plasma circulating cell-free DNA (cfDNA) the methylation of a panel of genes (FBLN1, HINT2, LAMC1, LTBP1, LTBP2, PSMA2, PSMA7, PXDN, TGFB1, UBE2L3, VIM and YWHAZ) and to evaluate the potential of these genes as novel HCC biomarkers in two different series, one from France and one from Thailand. Methylation in cfDNA was detected for FBLN1, PSMA7, PXDN and VIM, with substantial differences in methylation patterns between cases and controls for FBLN1 and VIM. Further, the average methylation level across analyzed CpG-sites was associated with higher odds of HCC for VIM (1.48 [1.02, 2.16] for French cases, and 2.18 [1.28, 3.72] for Thai cases), and lower odds of HCC for FBLN1 (0.89 [0.76, 1.03] for French cases and 0.75 [0.63, 0.88] for Thai cases). We also analyzed a set of HCC and adjacent tissues as well as The Cancer Genome Atlas’ (TCGA) data to further investigate the origin of methylation pattern in cfDNA and noted that the analysis of VIM could be improved based on the methylation pattern detected in TCGA data. Overall, our study provides evidence that changes in methylation levels of cfDNA could be used as potential cancer biomarkers and that VIM and FBLN1 methylation in cfDNA are associated with HCC and may represent useful plasma-based biomarkers for improved detection and patient surveillance.

Citation Format: Reetta Holmila, Athena Sklias, David C. Muller, Davide Degli Esposti, Paule Guilloreau, James Mckay, Suleeporn Sangrajrang, Petcharin Srivatanakul, Pierre Hainaut, Philippe Merle, Andre Nogueira da Costa, Zdenko Herceg. Targeted deep sequencing of plasma circulating cell-free DNA shows VIM and FBLN1 methylation as potential biomarkers for hepatocellular carcinoma. [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 4468.
Abstract 4488: Targeted sequencing revealed distinctive and pathogenic mutations in African Americans with colorectal cancer
Authors: Ashktorab Hassan , Azimi Hamed , Nickerson Mike , Bass Sara , Boland Joseph , Yeager Meredith , Varma Sudhir , Daremipouran Mohamed , Sherif Zaki , Ghavimi Shima , Shokrani Babak , Lee Edward , Laiyemo Adeyinka , Brim Hassan
Published: July 2016

Background & Aim: We recently analyzed 12 pairs of African American CRC tumors and their matched normal tissue using whole exome sequencing and established a panel of novel mutations that are potentially useful for the detection of CRC in this population. In this study, we examined APC, MSH3, and MSH6 mutations using targeted exome sequencing (TES) to determine distinctive mutations frequencies and their chronology in the neoplastic transformation.

Materials & Methods: A total of 140 colon samples: (30 normal, 21 adenomas, 33 advanced adenomas & 56 tumors collected from African Americans were used as our discovery set on an Ion Torrent platform. A subset of 36 samples were used as validation set on an Illumina platform. Bioinformatic analysis was performed on both sets of data. Common mutations were considered validated.

Results: Two novel MSH6 mutations were validated. Four known mutations in MSH3 were validated and were located in nonsynonymous (exon 10) and 1synonomous mutation (exon 18). As for the APC, 20 mutations were validated include 4 novel mutations. The novel mutations were 3 stopgain and 1 nonsynonymous located at the EB1 binding site, in the mutational cluster region (MCR), and at the 15 Amino Acid repeat.

Conclusion: We here defined novel mutations that target DNA MMR and APC genes in African Americans with colorectal lesions. Greater frequency of mutations in cells defective for DNA repair and APC genes is an advantage in cell growth and genetic instability and relevant to the initiating events of colon tumorigenesis.

Citation Format: Hassan Ashktorab, Hamed Azimi, Mike Nickerson, Sara Bass, Joseph Boland, Meredith Yeager, Sudhir Varma, Mohamed Daremipouran, Zaki Sherif, Shima Ghavimi, Babak Shokrani, Edward Lee, Adeyinka Laiyemo, Hassan Brim. Targeted sequencing revealed distinctive and pathogenic mutations in African Americans with colorectal 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 4488.
Abstract 4490: Comparisons of genetic alterations of breast cancer between East and West: Special emphases on young patients with ER+/HER2- tumors
Authors: Lin Ching-Hung , Lu Tzu-Pin , Huang Ruby Yun-Ju , Lu Yen-Shen , Lo Ko-Yun , Kuo Kuan-Ting , Chuang Eric Y. , Wu Pei-Fang , Chen I-Chun , Thiery Jean Paul , Huang Chiun-Sheng , Cheng Ann-Lii
Published: July 2016

A rapid surge of female breast cancer has been observed in young East Asians. As a first step toward understanding this phenomenon, we compared the genetic alterations between breast cancer form Taiwan (East) and Western countries (West). Under the same array platforms, the differences in gene copy number alterations (n = 120 in East and 1,948 in West) and gene expressions (n = 327 in East and 423 in West) were analyzed as further stratified by age and estrogen receptor (ER)/ HER2 status. Gene-gene interaction networks were explored by analyzing the overlapping gene with ingenuity pathway analysis. The ingenuity pathway analysis revealed the common differences between East and West in NFkB, PI3K, Akt, MAPK, ERK, IFNα, and Jnk networks. Among the patients <50 years with ER+/ HER2- tumors, we identified a uniquely higher frequency of the APOA1/C3/A4/A5 (the lipid metabolizing genes) gene cluster loss (29% vs. 12%, P = .005) and low expressions of these four genes (East/ West ratios ranged from .07 to .39) in East than that in West. We conclude that NFkB, PI3K, Akt, MAPK, ERK, IFNα, and Jnk networks are common differences between East and West, while APOA1/C3/A4/A5 loss and low expressions are unique genetic alterations for young Asian patients with ER+/ HER2- tumors.

Citation Format: Ching-Hung Lin, Tzu-Pin Lu, Ruby Yun-Ju Huang, Yen-Shen Lu, Ko-Yun Lo, Kuan-Ting Kuo, Eric Y. Chuang, Pei-Fang Wu, I-Chun Chen, Jean Paul Thiery, Chiun-Sheng Huang, Ann-Lii Cheng. Comparisons of genetic alterations of breast cancer between East and West: Special emphases on young patients with ER+/HER2- tumors. [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 4490.
Abstract 4495: Development and clinical application of an integrative genomic approach to personalized cancer therapy
Authors: Chen Rong
Published: July 2016

We implemented a personalized cancer therapy (PCT) program in a clinical setting, applying an integrative genomics approach to maximally elucidate the complexity of each tumor. For patients in the study, we performed whole exome sequencing and SNP microarrays on tumor and patient-matched normal samples, as well as RNA sequencing on available frozen samples, to identify somatic mutations, copy number alterations, gene fusions, and gene expression alterations in the tumor. Ion AmpliSeq Cancer Hotspot Panel v2 (CHPv2) was used to ensure high sensitivity in cancer mutation hotspots. Genomics results were integrated with cancer knowledge databases and for each cancer type a specific workflow was developed to optimize data interpretation. We returned genomics findings to 46 patients and their physicians, characterizing somatic alterations and predicting drug response, toxicity, and prognosis. Mean 17.3 cancer-relevant somatic mutations per patient were identified, 13.3-, 6.9-, and 4.7-fold more than could have been detected using CHPv2, Oncomine Cancer Panel (OCP), and FoundationOne, respectively. Our approach delineated the underlying genetic drivers at the pathway level and provided meaningful predictions of therapeutic efficacy and toxicity. Actionable alterations were found in 91% of patients (mean 4.9 per patient, including somatic mutations, copy number alterations, gene expression alterations, and germline variants), a 7.5-fold, 2.0-fold, and 1.9-fold increase over what could have been uncovered by CHPv2, OCP, and FoundationOne, respectively. The findings altered the course of treatment in 4 cases. These results show that a comprehensive, integrative genomic approach as outlined above significantly enhanced genomics-based personalized cancer therapy strategies.

Citation Format: Rong Chen. Development and clinical application of an integrative genomic approach to personalized cancer therapy. [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 4495.
Abstract 4526: Proscillaridin A effects on chromatin modifications and oncogene degradation in acute lymphoblastic leukemia
Authors: Armaos Gregory , Jacques-Ricard Simon , Da Costa Elodie , Beaudry Annie , Raynal. Noël J-M
Published: July 2016

Acute lymphoblastic leukemia (ALL) represents approximately 25% of all pediatric cancers diagnosed every year. In about 80% of cases, pediatric patients will attain an event-free 5-year survival. Unfortunately, patients who are resistant to treatment or who relapse have a poor prognosis. Hence, novel therapeutic approaches are necessary to increase survival rates. Epigenetic alterations, such as DNA methylation and histone modifications, are involved in disease development, progression, and in particular, resistance to treatment. These reversible alterations represent additional targets in ALL. Recently, we discovered candidate epigenetic drugs in FDA-approved drug libraries. We hypothesize that one drug in particular, the cardiac glycoside proscillaridin A, has both epigenetic and anti-cancerous properties in preclinical models of ALL and can therefore be repositioned for the treatment of the disease. To test our hypothesis, we treated two ALL cell lines Nalm-6 (pre-B ALL) and Molt-4 (T-ALL) in vitro with clinically relevant concentrations of proscillaridin A and analyzed cell growth, cell cycle, gene expression and chromatin modifications. We observed dose-dependent growth inhibition in all cell lines, with IC50 values of 3.0 and 2.3 nM in Nalm-6 and Molt-4, respectively. Our results using BrdU staining indicate a block in the G2/M phase of the cell cycle. By western blot, we detected a reduction in histone acetylation levels and in histone modifying enzymes CBP and Tip60, as well as the c-myc oncogene. These promising results illustrate the perspective of using the cardiac glycoside proscillaridin A as a novel epigenetic drug for the treatment of relapsed or refractory ALL.

Citation Format: Gregory Armaos, Simon Jacques-Ricard, Elodie Da Costa, Annie Beaudry, Noël J-M Raynal. Proscillaridin A effects on chromatin modifications and oncogene degradation in acute lymphoblastic leukemia. [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 4526.
Abstract 4530: EpiCapture: Benchmarking commercially available targeted bisulfite-sequencing platforms to gold-standard whole genome bisulfite sequencing
Authors: Tanic Miljana , Rodney Simon , Barrett James , Parker Hannah , Feber Andrew , Beck Stephan
Published: July 2016

Genomic DNA methylation is one of the most important epigenetic modifications in eukaryotes necessary for cellular differentiation and plays a crucial role in gene regulation of many oncogenes and tumor suppressor genes. Aberrant DNA methylation at the level of both individual gene promoters and on a genome-wide scale has been heavily implicated in cancer initiation and progression.

Whole genome bisulfite sequencing (WGBS) is the gold standard method for studying alterations in DNA methylation at a single base pare resolution. However, the high cost of WGBS is still prohibitive for high-throughput observational studies on large sample cohorts. Moreover, the WGBS data analysis requires substantial IT resources and a complex bioinformatics analysis. Targeted bisulfite sequencing enables a cost-effective and focused analysis of genomic regions of interest where most of DNA methylation alterations occur, such as promoters, CpG islands and shores, and enhancer regions. Currently, there are 2 main approaches for targeted bisulfite sequencing of medium-to-large genomic regions, reduced representation bisulfite sequencing (RRBS) and hybridization-based enrichment.

We have performed a comparison of 5 commercially available platforms for RRBS and in-solution hybridization-based enrichment panels. We analyzed a panel of two cancer cell lines (Hela and Coriell NA12878) and a normal blood reference gDNA sample, in duplicate on each platform using recommended sample inputs, and compared the results to WGBS data. Given that sample input requirements differ significantly between RRBS (∼100ng or less), and hybridization based methods (500ng - 1 μg), we have analyzed the normal blood reference gDNA sample at 500ng input in duplicate across all platforms. All samples were sequenced to a ∼30x depth on an Illumina HiSeq 2500 sequencer. We compared the genomic coverage, precision and off-target effects and overall concordance in methylome profiles for each method.

In summary, here we provide a comprehensive comparison of the performance of 5 different commercially available methods for medium to large scale targeted bisulfite sequencing, facilitating information to researchers for selection of appropriate method depending on required application.

Citation Format: Miljana Tanic, Simon Rodney, James Barrett, Hannah Parker, Andrew Feber, Stephan Beck. EpiCapture: Benchmarking commercially available targeted bisulfite-sequencing platforms to gold-standard whole genome bisulfite sequencing. [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 4530.
Abstract 4531: Dynamic long-range chromatin interaction controls expression of Il-21 in CD4+ T cells
Authors: Choi Yeeun
Published: July 2016

IL-21, a member of four-α-helical bundle type I cytokines, causes many pleiotropic effects, including terminal differentiation of B cells, immunoglobulin production, and the development of follicular helper T cells. IL-21 also promotes differentiation of Th17 cells and is implicated in the development of autoimmune disease. IL-21 can be strongly induced in CD4+ T cells by IL-6; however, very little is known about the mechanisms underlying the transcriptional regulation of the Il-21 gene at the chromatin level. Here, we identified insulator elements in the Il-21 locus with enhancer-blocking activity, which constitutively bind CTCF and cohesin. In naïve CD4+ T cells, these upstream and downstream CTCF-binding sites interact with each other to make a DNA loop; however, the Il-21 promoter does not interact with any cis-elements in the Il-21 locus. In contrast, stimulation of CD4+ T cells with IL-6 in the presence of αCD3 and αCD28 leads to recruitment of STAT3 and NFAT transcription factors to the promoter, as well as to novel distal enhancer region. This induces dynamic changes in chromatin configuration, bringing the promoter and the regulatory elements in close spatial proximity, facilitating IL-21 expression. The long-range interaction between the promoter and distal enhancer region was dependent on JAK/STAT3 signaling pathway but was disrupted in regulatory T cells, where IL-21 expression was repressed. In addition, we demonstrated that CTCF was required for optimal IL-21 expression and formation of chromatin looping in the Il-21 locus. Thus, our work uncovers a novel topological chromatin framework underlying proper transcriptional regulation of Il-21 gene.

Citation Format: Yeeun Choi. Dynamic long-range chromatin interaction controls expression of Il-21 in CD4+ T 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 4531.
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

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

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

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

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

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

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

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

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

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.
Abstract 4603: TCF7L1 constrains Wnt/β-catenin signaling and tumor growth in pancreatic cancer
Authors: Kershaw Kathleen M. , Arensman Michael D. , Ho Bridgette , Lay Anna R. , Dawson Nicole A. , Dawson David W.
Published: July 2016

Wnt/β-catenin signaling plays important roles in pancreatic adenocarcinoma (PDA) tumor initiation and progression through its promotion of various pro-tumorigenic phenotypes. While RNF43 mutations increase Wnt signaling activity and confer Wnt growth-dependency in a subset of PDA, other key driver mutations in the Wnt pathway are only rarely encountered in PDA. To gain additional insights into alternative mechanisms contributing to increased Wnt pathway activity in PDA, we examined the expression and activity of LEF/TCF family members, the key transcriptional binding partners for β-catenin in canonical Wnt signaling. Luciferase-based β-catenin-activated reporter (BAR) assays demonstrated variable Wnt activity across a large panel of PDA cell lines, which was used to dichotomize cell lines into groups with high or lower β-catenin/TCF-dependent transcription. Supervised analysis of PDA cell line microarray data and parallel western blot analyses identified distinct patterns of TCF/LEF expression, including increased TCF7 and TCF7L2 (aka TCF4) expression in high BAR lines and increased TCF7L1 (aka TCF3) expression in low BAR lines. RNAi-mediated knockdown of individual or combinations of TCF/LEFs in PDA lines revealed TCF7L1 and TCF7L2 both function to repress β-catenin-activated reporter activity. TCF7L1 protein was also found to be targeted for rapid downregulation following Wnt pathway activation by treatment with either Wnt3A ligand or GSK3β inhibitor. RNAi knockdown of TCF7L1 in PDA lines increased anchorage-independent growth and tumor cell migration in vitro and increased in vivo PDA growth in an orthotopic xenograft tumor model. In summary, differential patterns of TCF/LEF expression are correlated with and functionally responsible for differences in Wnt pathway activation and function in PDA. Furthermore, these data implicate abrogation of TCF7L1 growth suppressive phenotypes via β-catenin-mediated downregulation of TCF7L1 as a potentially important mechanism through which Wnt signaling promotes pancreatic tumorigenesis.

Citation Format: Kathleen M. Kershaw, Michael D. Arensman, Bridgette Ho, Anna R. Lay, Nicole A. Dawson, David W. Dawson. TCF7L1 constrains Wnt/β-catenin signaling and tumor growth in pancreatic 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 4603.
Abstract 4607: Mediator kinase module as a transducer of oncogenic Wnt/beta-catenin signaling
Authors: Clark Alison , Oldenbroek Marieke , Wang Yao , Spaeth Jason , Gao Fangjian , Jin Victor X. , Boyer Thomas
Published: July 2016

Dysregulation of Wnt/β-catenin signaling promotes colorectal cancer (CRC) and other types of malignancies through unprogrammed changes in gene transcription that drive tumorigenesis. CDK8 is a CRC oncoprotein whose amplification-dependent overexpression identifies a significant subset of CRC patients with poor prognosis. CDK8 kinase activity, along with Cyclin C (CycC), drives tumorigenesis by stimulating β-catenin transcriptional activity. Thus, inhibition of CDK8 kinase function offers a promising therapeutic approach for CDK8-overexpressing CRCs. CycC-CDK8, along with MED12 and MED13, compose a four-subunit “kinase” module within Mediator, a conserved multiprotein interface between gene-specific transcription factors and RNA Polymerase II. We have previously identified a network of physical and functional interactions within the Mediator kinase module critical for oncogenic Wnt/β-catenin signaling. Mechanistically, β-catenin binds directly to MED12 in Mediator, thus activating CycC-dependent CDK8 kinase activity and β-catenin transcriptional activity. More specifically, MED12-dependent CDK8 activation occurs through a direct interaction involving the MED12 N-terminus and a phylogenetically conserved surface groove on CycC. Here, we demonstrate that mutagenic disruption of the MED12/CycC interface in CRC cell lines leads to uncoupling of CycC-CDK8 to MED12 and core Mediator, and concomitant loss of Mediator-associated CDK8 activity. Transcriptome analysis of cells lacking CDK8 kinase function revealed downregulation of Wnt/β-catenin signaling and other oncogenic pathways, consequently impairing CRC cell proliferation and clonogenicity. Our studies therefore identify the MED12/CycC interface as a critical transducer of oncogenic Wnt/β-catenin signaling. By validating MED12/CycC as a potential therapeutic target, our findings have significant implications in current methods of treating CRC as they pave a way for development of novel, targeted inhibitors of Wnt/β-catenin signaling to treat colorectal and other CDK8-driven driven malignancies.

Supported by MH085320-05 (TGB), RP140435 (TGB) and T32DE14318 (ADC).

Citation Format: Alison Clark, Marieke Oldenbroek, Yao Wang, Jason Spaeth, Fangjian Gao, Victor X. Jin, Thomas Boyer. Mediator kinase module as a transducer of oncogenic Wnt/beta-catenin signaling. [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 4607.
Abstract 4623: Reevaluation of the role of Notch signaling in melanoma tumor development, melanoma cell survival and drug resistance
Authors: Mikheil Dareen , Rodriguez Carlos , Jayanthi Ashika , Yarlagadda Prithvi , Setaluri Vijaysaradhi
Published: July 2016

Notch is one of the simplest signaling pathways that is required for almost every cell type in the body. It plays several roles such as cell fate decision, differentiation and stem cell maintenance. Notch signaling plays a role in the maintenance of melanoblasts and melanocyte stem cells of the epidermis by preventing their apoptosis. Notch signaling has also been implicated in different aspects of melanoma biology including tumor initiation, progression and metastasis. Accordingly, Notch has been targeted for melanoma therapy both in preclinical xenograft models as well as clinical trials. Notch inhibitors, mainly γ-secretase inhibitors, have been widely used for this purpose. However, clinical trials with these agents have not been proven to be highly effective for melanoma treatment. In previously published studies we showed that Notch signaling is involved in regulation of melanoma trans-differentiation, a prognostic feature in primary melanoma. In this study, we reevaluated the role of Notch in melanoma development, cell survival and sensitivity to BRAF and MEK inhibitors. First, our preliminary data using the BRAFV600E/Pten mouse model showed that topical application of γ-secretase inhibitor DAPT delays tumor formation and growth. In several early passage metastatic human melanoma cell lines, we found that Notch receptors (Notch 1-3) are highly upregulated compared to normal epidermal melanocytes. Interestingly, mRNA levels for Notch1-3 receptors in normal melanocytes were either higher or comparable to their levels in the cell lines. This suggested that Notch receptor levels are tightly regulated in normal melanocytes by post-transcriptional mechanisms. Presence of cleaved Notch intracellular domain (NICD), both cytoplasmic and nuclear, in melanoma cells, but not in melanocytes, indicated constitutive activation of Notch signaling in melanoma cells. However, inhibition of Notch activation by treatment with γ-secretase inhibitor had no significant effect on survival of melanoma cells in vitro. Thus, Notch signaling appears to be dispensable for melanoma cell survival. Additionally, inhibition of Notch activation did not alter the sensitivity of melanoma cells to BRAF(V600E) and MEK inhibitors. Our results highlight the need for re-examination of Notch-regulated melanoma trans-differentiation as a therapeutic outcome.

Citation Format: Dareen Mikheil, Carlos Rodriguez, Ashika Jayanthi, Prithvi Yarlagadda, Vijaysaradhi Setaluri. Reevaluation of the role of Notch signaling in melanoma tumor development, melanoma cell survival and drug resistance. [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 4623.