bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3322: Spatio-temporal regulation of epithelial transformation by mistrafficking of EGFR ligand, epiregulin
Authors: Singh Bhuminder , Bogatcheva Galina , Washington Mary K. , Coffey Robert J.
Published: May 2015

The EGF receptor (EGFR) ligand epiregulin (EREG) is delivered preferentially to the basolateral cell surface of polarized MDCK cells. Recently, we showed that EREG basolateral trafficking is regulated by a conserved tyrosine residue within a YXXΦ motif (Y156ERV) in its cytoplasmic domain. Interestingly, a Y156A substitution led to apical mistrafficking of EREG and transformation of polarized MDCK cells (PNAS 110: 8960-5, 2013). We have identified EREG mutations (R147stop) in human tumors that would disrupt the basolateral sorting motif of EREG and now report that EREG harboring these patient mutations mistrafficks to the apical surface. We propose that ligand mistrafficking is a driver of transformation rather than being a mere passenger. To test our proposal wild-type and mutant EREG will be inducibly (Tet-ON) expressed at various stages of MDCK cyst formation and maintenance in 3D Matrigel cultures where we have now recapitulated the transformation phenotype induced by EREG-mistrafficking. Results from these experiments will be presented at the meeting.

Citation Format: Bhuminder Singh, Galina Bogatcheva, Mary K. Washington, Robert J. Coffey. Spatio-temporal regulation of epithelial transformation by mistrafficking of EGFR ligand, epiregulin. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3322. doi:10.1158/1538-7445.AM2014-3322
Abstract 3325: Role of AP2A1 in EGFR nuclear translocation and transcriptional activation activity
Authors: Chen Huai-Gu , Hsu Sheng-Chieh
Published: May 2015

The tyrosine kinase activity and endocytosis of epidermal growth factor receptor (EGFR) are activated by ligand binding. The adaptor protein 2 (AP2) complex found in the clathrin coated vesicles mediates intracellular trafficking of EGFR. Accumulating evidence suggest that the nucleus is one of intracellular destination. Nuclear EGFR severs as a transcriptional co-activator to regulate expression of several genes which are involved in tumor growth, progression and drug resistance. The carboxyl terminal 186 amino acids of EGFR has transcriptional activation activity and the AT-rich sequence (ATRS) of the target gene promoter is required for EGFR binding. In the present study, we show that AP2A1, the alpha 1 adaptin subunit of AP2 complex, interact with EGFR not only in the cytoplasm but also in the nucleus. The EGFR-AP2A1 interaction is stimulated by EGF and suppressed by Iressa, a tyrosine kinase inhibitor. Knockdown AP2A1 expression in cancer cells abrogates EGF-induced EGFR nuclear translocation. Interesting, the ATRS reporter activity is enhanced by depletion of cellular AP2A1. These results indicate that AP2A1 is required for EGFR nuclear translocation and suppresses the transcriptional activation activity of EGFR. Thus, we demonstrate here the novel role of AP2A1 in EGFR trafficking and transcriptional regulation.

Citation Format: Huai-Gu Chen, Sheng-Chieh Hsu. Role of AP2A1 in EGFR nuclear translocation and transcriptional activation activity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3325. doi:10.1158/1538-7445.AM2014-3325
Abstract 3352: miR-122 targets pyruvate kinase M2 and affects metabolism of hepatocellular carcinoma
Authors: Xu Zhi , Liu Angela , Lee Nikki , Chen Jinfei , Luk John M.
Published: May 2015

In contrast to normal differentiated cells that depend on mitochondrial oxidative phosphorylation for energy production, cancer cells have evolved to utilize aerobic glycolysis (Warburg's effect), with benefit of providing intermediates for biomass production. MicroRNA-122 (miR-122) is highly expressed in normal liver tissue regulating a wide variety of biological processes including cellular metabolism, but is reduced in hepatocellular carcinoma (HCC). Overexpression of miR-122 was shown to inhibit cancer cell proliferation, metastasis, and increase chemosensitivity, but its functions in cancer metabolism remains unknown. The present study aims to identify the miR-122 targeted genes and to investigate the associated regulatory mechanisms in HCC metabolism. We found the ectopic overexpression of miR-122 affected metabolic activities of HCC cells, evidenced by the reduced lactate production and increased oxygen consumption. Integrated gene expression analysis in a cohort of 94 HCC tissues revealed miR-122 level tightly associated with a battery of glycolytic genes, in which pyruvate kinase (PK) gene showed the strongest anti-correlation coefficient (Pearson r = -0.6938, p = <0.0001). In addition, reduced PK level was significantly associated with poor clinical outcomes of HCC patients. We found isoform M2 (PKM2) is the dominant form highly expressed in HCC and is a direct target of miR-122, as overexpression of miR-122 reduced both the mRNA and protein levels of PKM2, whereas PKM2 re-expression abrogated the miR-122-mediated glycolytic activities. The present study demonstrated the regulatory role of miR-122 on PKM2 in HCC, having an implication of therapeutic intervention targeting cancer metabolic pathways.

Citation Format: Zhi Xu, Angela Liu, Nikki Lee, Jinfei Chen, John M. Luk. miR-122 targets pyruvate kinase M2 and affects metabolism of hepatocellular carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3352. doi:10.1158/1538-7445.AM2014-3352
Abstract 3360: Loss of sulf-1 reprograms energy metabolism in ovarian cancer
Authors: Mondal Susmita , Roy Debarshi , Pereira Juliana Camacho , Chini Eduardo N. , Shridhar Vijayalakshmi
Published: May 2015

We have previously shown that loss of HSulf-1, an endosulfatase that removes 6-O sulfation of HSPGs augmented heparin binding growth factor (HB-GF) signaling resulting in enhanced tumorigenesis, angiogenesis and metastasis in ovarian cancer. However, the role of HSulf-1 in energy metabolism and how the increased growth factor signaling in cells with loss of HSulf-1 altered cellular metabolism in cancer yet to be investigated. Here, we show for the first time that batch clonal lines of HSulf-1 knockdown cells [OV202 (Sh1/2)] and HSulf-1 knock out (KO) MEFs showed a significantly higher glycolytic flux (glucose uptake and lactate production) and reduced oxygen consumption rate (OCR) compared to a non-transduced control (OV202NTC) and wild-type (WT) MEF cells respectively. Conversely, enhanced expression of HSulf-1 in TOV21G cells resulted in lower glycolytic flux and higher OCR. Microarray analysis revealed that several of the genes in the glycolytic pathway such as Glut1, HK2, PFKFB3, ALDOC, PGAM, PKM2 and LDHA were upregulated in Sh1/2, cells compared to NTC cells. Expression of these genes and their respective products were validated both at the mRNA and protein levels in OV202Sh1/2, TOV21G Vec and MEFs KO cells compared to OV202NTC, TOV21G Cl13 and MEF WT respectively. Importantly, we found that loss of HSulf-1 decouples the energy flow from glycolysis to mitochondria by lowering the PDH and up-regulating PDK1 expression. We further demonstrated that loss of HSulf-1 reduced the total cellular ATP levels in Sh1/Sh2, TOV21G VEC, and MEFs KO cells, all consistent with lower mitochondrial activity as evidenced by decreased total number of mitochondria, its cross sectional area and perimeter in Sh1/Sh2, TOV21G Vec and MEF KO cells. This augmented aerobic glycolysis with loss of HSulf-1 was further corroborated by metabolomics study which showed significantly higher glycolytic metabolites such as G-6P, F1,6BP, F2,6BP, 3PG. While most of the TCA intermediates were downregulated in Sh1 and Sh2 cells; glutamate, glutamine, α-ketoglutarate and 2-hydroxyglutarate were higher upon loss of HSulf-1. These results suggests that loss of HSulf-1 plays a pivotal role in reprogramming the energy metabolism towards the Warburg effect which are critical to sustain cancer cells proliferation, transformation and angiogenesis.

Citation Format: Susmita Mondal, Debarshi Roy, Juliana Camacho Pereira, Eduardo N. Chini, Vijayalakshmi Shridhar. Loss of sulf-1 reprograms energy metabolism in ovarian cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3360. doi:10.1158/1538-7445.AM2014-3360
Abstract 337: Carfilzomib synergistically enhances ABT-263-induced apoptosis due to NOXA induction in KRAS wild type and mutant colorectal cancer cell lines
Authors: Huang Shengbing , Okamoto Koichi , Sinicrope Frank A.
Published: May 2015

BACKGROUND. Proteosome inhibition is associated with induction of the proapoptotic BH3 protein NOXA. Therefore, we hypothesize that the combination of the irreversible proteosome inhibitor, carfilzomib, will cooperatively enhance apoptosis induction by the BH3 mimetic drug, ABT-263. Given that nononcogenic addiction to proteosome activity has been reported in KRAS mutant cells, we utilized isogenic colorectal cancer (CRC) cell lines in which the endogenous wild-type (wt) or mutant alleles had been inactivated through targeted homologous recombination. Since proteasome blockade results in proteotoxic stress, we determined whether an autophagy inhibitor can enhance chemosensitivity.

EXPERIMENTAL METHODS. Isogenic KRAS mutant (MUT) CRC cells (HCT116, DLD1; obtained from B. Vogelstein, JHU) were incubated with carfilzomib (25,50nM) alone or combined with ABT-263 (1 µM) and apoptosis was measured by caspase cleavage and annexin V+ labeling. Synergy was measured by calculation of the combination index. Effect of carfilzomib on Bcl-2 family proteins was determined by immunoblotting. Interaction of NOXA with MCL-1 was examined by immunoprecipitation. Knockdown of NOXA was achieved by lentiviral shRNA. Cells were incubated with drugs alone or combined with chloroquine, and autophagy (LC3I/II conversion) and apoptosis were analyzed.

RESULTS. Carfilzomib was shown to potently and synergistically enhance ABT-263-induced apoptosis (2-fold) in both KRAS MUT and wild type (WT) cell lines, with the effect being modestly higher in wild type cells, as shown by annexin V+ staining and caspase cleavage. Carfilzomib induced NOXA expression in both KRAS MUT and WT cells, and reduced MCL-1 expression. Induced NOXA was shown to bind and sequester MCL-1 proteins. Knockdown of NOXA attenuated drug-induced apoptosis. Autophagy inhibition by chloroquine accumulated LC3II, but only modestly increased carfilzomib-induced cell death.

CONCLUSION. Irreversible proteasome inhibition by carfilzomib synergistically enhanced ABT-263-induced cell death that is mediated by NOXA upregulation and its sequestration/neutralization of MCL-1. The finding of similar efficacy in KRAS MUT vs WT cells suggests that this combination may represent a novel therapeutic strategy in this tumor subset.

Citation Format: Shengbing Huang, Koichi Okamoto, Frank A. Sinicrope. Carfilzomib synergistically enhances ABT-263-induced apoptosis due to NOXA induction in KRAS wild type and mutant colorectal cancer cell lines. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 337. doi:10.1158/1538-7445.AM2014-337
Abstract 3372: Constraints-based metabolic flux analysis approach links tumor stage to metabolic adaptations and survival in cancer cells
Authors: Achreja Abhinav , Yang Lifeng , Zhao Hongyun , Marini Juan , Nagrath Deepak
Published: May 2015

The Warburg effect has been observed in many cancers and their high glycolytic capacity has signified their dependence on glucose. More recently, glutamine has emerged not only as an important nutrient for many cancers, but also necessary for their elevated energetic requirements. Due to these high energetic demands, certain cancer cells become addicted to glutamine to maintain viability. We postulate that distinct metabolic reconfigurations of certain cancers define their dependence or independence on glutamine for survival while maintaining their proliferative propensity and redox status. An intricate picture of the metabolic profiles is to be drawn from estimating intracellular fluxes, by combining stable isotope tracer measurements and experimental metabolomics data from different cancer cell lines, which have been observed to be glutamine dependent and independent. To this extent, we describe an approach that utilizes a redox-balanced model incorporating the electron transport chain and comprehensive amino-acid metabolic reactions to elucidate the importance of oxidative phosphorylation, often overlooked in classical approaches. Diving deeper into the foray of metabolic reprogramming, we perform in silico experiments using a constraint-based multi-objective modeling approach. This methodology elucidates the switching of metabolic pathways in glutamine-dependent and -independent cancers under nutrient-available and nutrient-deprived conditions. Our approach assumes that cancer cells operate at optimal levels, maintaining multiple objectives under certain environmental conditions. Constraining the proliferative phenotype from a maximal to minimal levels of the cells under different nutrient conditions emulates the observed behavior of glutamine-dependent cells under deprivation conditions and contrasts their metabolic reprogramming against that of glutamine-independent cells. We corroborated our simulations with experimentally derived metabolic fluxes and found that glutamine anabolism over catabolism dictates adaptations and survival in invasive cancers. Our results will lead to identification of potential targets for inducing nutrient-sensitivity and enhance current therapeutic approaches.

Citation Format: Abhinav Achreja, Lifeng Yang, Hongyun Zhao, Juan Marini, Deepak Nagrath. Constraints-based metabolic flux analysis approach links tumor stage to metabolic adaptations and survival in cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3372. doi:10.1158/1538-7445.AM2014-3372
Abstract 3393: Nuclear receptor Nurr1 mediates cell proliferation and is involved in a p53-microRNA-34 regulatory network
Authors: Beard Jordan A. , Hills Justin L. , Tenga Alexa , Takwi Apana A. , Chen Taosheng
Published: May 2015

Nuclear receptor (NR) subfamily 4 member 2 (NR4A2), also known as Nurr1, has been shown to have oncogenic properties, in which it can mediate cell transformation, migration and drug resistance. However, the manner in which Nurr1 mediates these cancer-promoting features is not completely understood, but research implicates p53 as a potential intermediary. In this study, we show that Nurr1 enhances the proliferative rate of colorectal cancer cells. This increase in cell growth is accompanied by an upregulation of cyclin D3, a putative target gene of Nurr1. To understand how Nurr1 itself is regulated, we performed a reporter screen to identify microRNA regulators of Nurr1's 3’UTR. We identified miR-34c as a putative regulator of Nurr1 and this direct regulation was confirmed through site-directed mutagenesis of the predicted seed region. We next determined if p53 activation could affect Nurr1 expression through transcriptional activation of miR-34. Using Nutlin-3a to activate p53, we found that Nurr1 expression decreased in a dose response manner. Lastly, we sought to determine if Nurr1 could attenuate p53 activation. We found that overexpression of Nurr1 in colorectal cancer cells caused an attenuation of p53 transactivation of target genes following Nutlin-3a treatment. Our results suggest that a p53-miR-34-Nurr1 regulatory network exists, which would allow p53 to maintain Nurr1 in a suppressed state through miR-34-mediated regulation and prevent tumorigenesis. In cases of p53 mutation or Nurr1 overexpression, this network would be compromised, allowing Nurr1 to enhance tumorigenesis or drug resistance.

Citation Format: Jordan A. Beard, Justin L. Hills, Alexa Tenga, Apana A. Takwi, Taosheng Chen. Nuclear receptor Nurr1 mediates cell proliferation and is involved in a p53-microRNA-34 regulatory network. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3393. doi:10.1158/1538-7445.AM2014-3393
Abstract 3396: Dual lineage inhibition of ETV1 and KIT disrupts the ETV1-KIT feed forward circuit and potentiates imatinib antitumor effect in GIST oncogenesis
Authors: Ran Leili , Sirota Inna , Cao Zhen , Murphy Devan , Shukla Shipra , Rossi Ferdinando , Wongvipat John , Tap William D. , Besmer Peter , Antonescu Cristina R. , Chen Yu , Chi Ping
Published: May 2015

Gastrointestinal stromal tumour (GIST) is one of the most common types of human sarcoma and is primarily defined by activating mutations in the KIT or PDGFRA receptor tyrosine kinases. Despite the initial clinical success of imatinib that specifically target mutant KIT and PDGFRA, imatinib resistance has become the biggest challenge in the management of advanced GIST patient. Novel therapeutics that can improve the efficacy of first line imatinib therapy and/or prevent and overcome imatinib resistance is imperative. The ETS family member, ETV1, has been previously identified as a lineage-specific survival factor that cooperates with mutant KIT by ETV1 protein stabilization by active MAP kinase signaling downstream of KIT signaling in GIST oncogenesis. Here we demonstrate that ETV1 is required for GIST initiation and maintenance in vivo using compound genetically engineered mouse models (GEMM). We further identified that ETV1 forms a feed forward circuit in GIST oncogenesis where the ETV1 protein is stabilized by active MAP kinase signaling downstream of KIT and stabilized ETV1enhances KIT expression through direct binding to the KIT enhancer regions in both GIST mouse models and human GIST cell lines. The dual lineage targeting of KIT by imatinib and ETV1 by MEK162 (an MEK inhibitor) disrupts the ETV1-KIT feed forward circuit and induces more apoptosis than single agent imatinib or MEK162 in human GIST cells. The combination therapy resulted in complete tumor regression whereas single agent imatinib or MEK162 resulted in stabilization of disease in human GIST xenograft studies. Moreover, the combination therapy also induced more tumor fibrosis than single agent imatinib or MEK162 in GIST GEMM. These observations demonstrate the in vivo role of ETV1 in GIST oncogenesis and the feasibility of targeting ETV1 protein stability by inhibiting MAP kinase signaling. They also suggest that the dual lineage targeting of ETV1 and KIT by the combination therapy may provide a more effective therapeutic strategy than imatinib alone in GIST management.

Citation Format: Leili Ran, Inna Sirota, Zhen Cao, Devan Murphy, Shipra Shukla, Ferdinando Rossi, John Wongvipat, William D. Tap, Peter Besmer, Cristina R. Antonescu, Yu Chen, Ping Chi. Dual lineage inhibition of ETV1 and KIT disrupts the ETV1-KIT feed forward circuit and potentiates imatinib antitumor effect in GIST oncogenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3396. doi:10.1158/1538-7445.AM2014-3396
Abstract 34: The receptor for hyaluronan acid mediator motility (RHAMM, CD168) expression in EMT-like cancer cells is a predictor of tumor progression in colorectal cancer
Authors: Mele Valentina , Koelzer Viktor H. , Huber Bettina , Muraro Manuele G. , Pfaff Dennis , Iezzi Giandomenica , Terracciano Luigi M. , Karamitopoulou Eva , Zlobec Inti , Lugli Alessandro
Published: May 2015

Background/Aim: The receptor for hyaluronan acid mediator motility (RHAMM) is a marker of tumor progression and decreased overall survival in patients with different solid tumor types. The aim of the present study was to investigate the functional and clinical role of RHAMM in colorectal cancer (CRC) using in vitro and in vivo models and to validate the results on a cohort (n=200) of CRC patients with full clinico-pathological features.

Material and Methods: Two established CRC cell lines HT29 and HT116 (mismatch-repair proficient and deficient) expressing RHAMM by flow-cytometry were selected for proliferation, invasion and migration assays. For the in vivo analysis tumor cells were injected in immune-deficient NOD/SCID mice. RHAMM expression was additionally analyzed by immunohistochemistry on 200 CRC whole tissue sections, with particular focus on the single, detached and aggressive EMT-like cells at the invasion front and correlated to clinicopathological data and survival.

Results: In vitro, RHAMM silenced HT29 and HCT116 cell lines showed a significant decrease in invasiveness and migration compared to untreated cells. In vivo, a lower tumorigenicity of HT29 and HT116 cells silenced for RHAMM compared to untreated cells was reflected by smaller tumor volumes in the injected immune-deficient NOD/SCID mice. In the immunohistochemical analysis RHAMM expression in EMT-like cancer cells was associated with lymphatic invasion (p=0.0013), higher tumor grade (p=0.021) and worse survival (p=0.034).

Conclusion: In vitro and in vivo findings as well as results on clinical patient data support the notion that over-expression of RHAMM is involved in tumor progression in CRC and highlight a promising basis for a therapeutic approach.

Citation Format: Valentina Mele, Viktor H. Koelzer, Bettina Huber, Manuele G. Muraro, Dennis Pfaff, Giandomenica Iezzi, Luigi M. Terracciano, Eva Karamitopoulou, Inti Zlobec, Alessandro Lugli. The receptor for hyaluronan acid mediator motility (RHAMM, CD168) expression in EMT-like cancer cells is a predictor of tumor progression in colorectal cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 34. doi:10.1158/1538-7445.AM2014-34
Abstract 3406: Activity of catechol-O-methyltransferase gene promoter polymorphisms in renal cancer cells
Authors: Chang Inik , Fukuhara Shinichiro , Wong Darryn K. , Gill Ankurpreet , Shahryari Varahram , Yamamura Soichiro , Majid Shahana , Saini Sharanjot , Hirata Hiroshi , Ueno Koji , Deng Guoren , Dahiya Rajvir , Tanaka Yuichiro
Published: May 2015

Catechol-compounds such as 4-hydroxy-estrogen have been shown to play a role in the renal carcinogenesis process. To counter the effects of the highly reactive and mutagenic catechol-estrogens, the catechol-O-methyltransferase (COMT) gene detoxifies these compounds by forming methoxy compounds, which have been shown to be biologically inactive. Proper COMT function is thus vital for cells and a mechanism by which activity levels can be affected is through polymorphisms. The Val(158)Met polymorphism that correlates with renal cancer has been shown to reduce COMT enzymatic activity, but also of importance are those variants in the promoter region which can lead to alterations in gene expression levels. The aim of this study was to determine if polymorphisms of the COMT promoter can influence their transcription levels. To examine this, plasmids containing wildtype regions of the COMT promoter were obtained and site-directed mutagenesis used to create polymorphic variants. Constructs were then transfected into the Caki-1 renal cell line to measure their effects on gene expression by using a luciferase reporter assay. A total of 8 polymorphic sites covering the distal and proximal promoter region were evaluated. Results indicate that the variant at polymorphic sites rs2020917 (C to T), rs13306278 (C to T), and rs6269 (A to G) caused a significant reduction (P<0.01) whereas rs165656 (G to C) and rs165722 (C to T) caused a significant increase (P<0.01) in luciferase activity. No differences were observed for rs737865 (A to G), 4646312 (T to C), and rs3810595 (G to C). These results demonstrate polymorphisms of the COMT promoter region to cause altered expression levels in renal cells and warrant further studies with clinical case-control specimens to determine their associations for renal cell cancer.

Citation Format: Inik Chang, Shinichiro Fukuhara, Darryn K. Wong, Ankurpreet Gill, Varahram Shahryari, Soichiro Yamamura, Shahana Majid, Sharanjot Saini, Hiroshi Hirata, Koji Ueno, Guoren Deng, Rajvir Dahiya, Yuichiro Tanaka. Activity of catechol-O-methyltransferase gene promoter polymorphisms in renal cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3406. doi:10.1158/1538-7445.AM2014-3406
Abstract 3423: The genomic evolution of prostate cancer under the selective pressure of anti-androgen therapy
Authors: Gsponer Joël Roman , Dietsche Tanja , Rufle Alexander , Lorber Thomas , Juskevicius Darius , Perrina Valeria , Lenkiewicz Elisabeth , Zellweger Tobias , Bachmann Alexander , Barrett Michael T. , Rentsch Cyrill A. , Ruiz Christian , Bubendorf Lukas
Published: May 2015

Background

The implementation of novel technologies such as array comparative genomic hybridization (aCGH) and next-generation sequencing has led to a deeper understanding of the genomic nature of cancer. However, these analyses have classically been done without respecting intra-tumor heterogeneity. Here, we applied a methodology that allows us analyzing the genomic profile of distinct tumor populations from individual tumors and their clonal evolution during the progression to castration-resistant disease, thus enabling the discovery of novel genetic alterations in the course of the development of therapy resistance.

Methods

Matched pre- and post- hormone treated fresh frozen and/or formalin fixed prostate cancer samples were selected from our biobank. Clonal tumor populations were flow-sorted according to their nuclear DNA content. Sorted tumor populations were subjected to whole genome CGH and to full exome sequencing analyses by the use of Agilent SurePrint 2x400k microarrays and the SureSelect All Exon Kit, respectively. Target aberrations were evaluated using a prostate cancer tissue micro array (TMA) and fluorescence in situ hybridization as well as immunohistochemistry.

Results

The de-convolution of the genomic evolution in the investigated tumors shows that genetic aberrations and mutations are newly occurring under the selective pressure of anti-androgen therapy. More importantly, judging on the temporal dynamics of changes in allelic frequencies, we cannot only observe events that become more abundant, but also cancer associated mutations that are negatively selected for. We are currently following up several of these targets, of which at least one shows significant association with overall survival and differences in its expression pattern between hormone naïve and castration resistant cancer.

Conclusions

Genomic profiling of distinct clonal tumor populations during prostate cancer progression allows for analysis of intra-tumoral heterogeneity and the underlying clonal evolution. Importantly, this approach identifies genomic aberrations that were selected for under the pressure of hormone ablation therapy.

Citation Format: Joël Roman Gsponer, Tanja Dietsche, Alexander Rufle, Thomas Lorber, Darius Juskevicius, Valeria Perrina, Elisabeth Lenkiewicz, Tobias Zellweger, Alexander Bachmann, Michael T. Barrett, Cyrill A. Rentsch, Christian Ruiz, Lukas Bubendorf. The genomic evolution of prostate cancer under the selective pressure of anti-androgen therapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3423. doi:10.1158/1538-7445.AM2014-3423
Abstract 3431: Functional characterization of ARID2 in melanoma and melanocytes
Authors: Luo Flora , Garraway Levi
Published: May 2015

In a recent exome sequencing study conducted by our laboratory, AT-rich interaction 2 (ARID2) was found to be mutated in 9% of melanoma samples.  Of all the novel melanoma genes identified, ARID2 had the highest loss of function mutation burden—suggesting that ARID2 is likely inactivated in melanoma.  Although it is well-established that ARID2 is a member of the ATP-dependent SWI/SNF chromatin remodeling complex, the mechanisms by which ARID2 functions as a tumor suppressor remain unclear, and its role in melanoma is completely unknown.  The goal of this project is to characterize the role of ARID2 in melanoma genesis.  Our preliminary data indicates that ARID2-mutant melanoma cells express low levels of both ARID2 and MITF—the master regulator of melanocyte differentiation and a lineage oncogene.   On the other hand, other SWI/SNF components appear necessary for survival of an ARID2-mutant cell line. Ongoing efforts in the laboratory include soft agar assays and mouse xenografts to determine whether knockout of ARID2 via CRISPR technology can transform immortalized primary melanocytes.  We are also investigating the larger SWI/SNF dependency of these mutant cell lines.  By studying ARID2 inactivation in melanoma, we ultimately seek to pinpoint targetable vulnerabilities of ARID2-mutant cells from many cancer types.

Citation Format: Flora Luo, Levi Garraway. Functional characterization of ARID2 in melanoma and melanocytes. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3431. doi:10.1158/1538-7445.AM2014-3431
Abstract 346: Differentiation induced apoptosis in AML cells: The role of p73 in p53-independent versus p53-mediated apoptosis
Authors: Roberts Michael , Saffie Rizwan , Salmons Harold , Ghoto Mansoor , Schneider Juliana , Forrester Jeffrey
Published: May 2015

Human acute myeloid cell lines can be induced to differentiate into macrophage-like cells by phorbol esters. Differentiation is accompanied by cessation of cell proliferation and initiation of apoptosis. Many of these cell lines, like HL-60, are p53 deficient and therefore execute a differentiation-induced, p53-independent program of cell death. Evaluating changes in gene expression during a time course of PMA-induced HL-60 cell differentiation by DNA microarray analyses and RT-qPCR reveals up-regulation of several pro-apoptotic genes (ex. TNF, MCL1, EGR2, BCL3) and down-regulation of several anti-apoptotic genes (ex. BCL2, ANXA3, TSC22D3). Interestingly, RNA levels of p73 are unchanged, suggesting p73 does not substitute for the missing p53 function via activation of p73 gene transcription, however, we report here that p73 protein levels increase dramatically during the differentiation time course. By reintroducing p53 into HL-60 cells, we compare the changing gene expression profile during p53-mediated apoptosis versus that which occurs during phorbol ester-induced p73 accumulation through protein stabilization. Common and unique gene expression changes define the putative p73-dependent apoptosis program compared to p53-dependent apoptosis.

Citation Format: Michael Roberts, Rizwan Saffie, Harold Salmons, Mansoor Ghoto, Juliana Schneider, Jeffrey Forrester. Differentiation induced apoptosis in AML cells: The role of p73 in p53-independent versus p53-mediated apoptosis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 346. doi:10.1158/1538-7445.AM2014-346
Abstract 3483: A centrifugal ultrafiltration-based method for rapid purification of exosomes from biological samples
Authors: Cappione Amedeo , Gutierrez Sara , Mabuchi Masaharu , Smith Janet , Strug Ivona , Nadler Timothy
Published: May 2015

Exosomes represent a subset of small particles secreted by many types of cells under both normal and pathological conditions. The release of microvesicles has demonstrated biological relevance; these particles act as mediators of intercellular communication both within the local microenvironment of the release site as well as systemically. Moreover, since exosomes contain RNA (messenger and miRNA) and protein (membrane-bound and cytosolic) from their cells of origin, and given that this content can be influenced by cell state, they also potentially represent a burgeoning target for biomarker discovery. Critical to understanding the physiological significance of these particles is the development of preparative techniques permitting reliable isolation of purified fractions. While numerous methods of exosome purification exist, including ultracentrifugation, immunoaffinity-based isolation using magnetic beads, precipitation, and ultrafiltration, most are plagued by sample limitations or require long and tedious workflows to achieve success. Here we present a rapid alternative method for the selective fractionation of exosomes from biological samples using an ultrafiltration device. Since the method is spin-based and dependent on size exclusion, the device has broad applications with regards to sample volume and/or type. Optimization of the protocol was aided through use of a mid infrared (MIR)-based spectroscopy platform that permits simultaneous monitoring of lysis conditions, protein quantitation, and analysis of total lipid content during exosome fractionation. Given the ultrafiltration device's capacity for buffer exchange and sample concentration, purified fractions can be easily formatted to meet the requirements of any downstream analysis platform. To demonstrate this, resulting fractions were assayed by numerous techniques including flow cytometry, western blotting, ELISA-based assays, and electron microscopy.

Citation Format: Amedeo Cappione, Sara Gutierrez, Masaharu Mabuchi, Janet Smith, Ivona Strug, Timothy Nadler. A centrifugal ultrafiltration-based method for rapid purification of exosomes from biological samples. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3483. doi:10.1158/1538-7445.AM2014-3483
Abstract 3485: Efficient transfection of cancer cell lines using the 4D-NucleofectorTM System
Authors: Schroeder Jenny , Altrogge Ludger , Lorbach Elke , Kokatam Srinivasan , Schaepermeier Sabine , Weigel Meike , Andretta-Beu Gina , Buesch Stefanie , Grabeck Tamara , Krumnow Alexandra , Spicker Sonja , Kallol Sampada , Kapoor Preeti , Toell Andrea
Published: May 2015

Cell lines isolated from tumors are an important tool to study cancer in vitro. They can be used for drug development as well as for understanding the basic mechanisms underlying cancer. Transfection of cancer cell lines with different molecules like plasmid DNA, siRNA or mRNA is often an integral part for this kind of research.

Lonza's 4D-Nucleofector™ System is a modular system for the efficient transfection of primary cells and cell lines with a variety of substrates including plasmid DNA and siRNA. The 4D-Nucleofector™ X Unit supports the Nucleofection™ in two different formats. The aluminum-free 20µl Nucleocuvette™ Strip allows the transfection of low cell numbers down to 2x104 cells per reaction. As 16 reactions can be performed in parallel it is well suited for optimizing Nucleofection™ Conditions for cells lacking a ready-to-use Optimized Protocol. For higher cell numbers of up to 2x107 cells per reaction the same Nucleofection™ Conditions can be applied in the 100μl single Nucleocuvette™ Vessel. For higher throughput needs the 96-well Shuttle™ Add-on can be connected to the 4D-Nucleofector™ System. With this add-on six 20µl Nucleocuvette™ Strips can be processed in parallel allowing for screening applications or accelerating the optimization of transfection parameters for many cell types.

In this study we used the 4D-Nucleofector™ System in combination with the 96-well Shuttle™ Add-on for optimizing transfection conditions for multiple cancer cell lines. An exemplary optimization process is depicted for the human prostate carcinoma cell line DU 145 and for the human colorectal adenocarcinoma cell line COLO 205. Optimization steps included the selection of the appropriate Nucleofector™ Solution, Nucleofector™ Program and plasmid DNA concentration.

Transfection parameters were thereby optimized for a variety of adherent and non-adherent human cancer cell lines resulting in transfection efficiencies of up to 99%; while maintaining high cell viability.

Citation Format: Jenny Schroeder, Ludger Altrogge, Elke Lorbach, Srinivasan Kokatam, Sabine Schaepermeier, Meike Weigel, Gina Andretta-Beu, Stefanie Buesch, Tamara Grabeck, Alexandra Krumnow, Sonja Spicker, Sampada Kallol, Preeti Kapoor, Andrea Toell. Efficient transfection of cancer cell lines using the 4D-NucleofectorTM System. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3485. doi:10.1158/1538-7445.AM2014-3485
Abstract 3499: Funding innovative technologies for cancer research at the National Cancer Institute
Authors: Dickherber Anthony
Published: May 2015

In the fall of 2013, the National Cancer Institute re-launched the Innovative Molecular Analysis Technologies (IMAT) program, committing up to $10.5 million through unique funding mechanisms (3 year R21s and R33s) to better support investigators through both the early and advanced stages of technology development over traditional funding mechanisms for exploratory research. The IMAT program has supported the development of various analytical and sample processing platforms that have made substantial contributions across the cancer research continuum, and the process of winning an award remains highly competitive. A variety of IMAT-supported research projects will be highlighted to demonstrate the variety and high level of innovation evident in the IMAT portfolio of supported research. An emphasis will be placed on recently completed projects so that investigators interested in next generation tools may become aware of possible developments through NCI-funded projects for their research. In addition, trends from recently awarded projects will be described to provide insight for potential applicants on the nature of the program and the competition. Potential projects of interest and identified technology gaps will also be discussed. The IMAT program continues to represent a unique resource for highly innovative technology development.

Citation Format: Anthony Dickherber. Funding innovative technologies for cancer research at the National Cancer Institute. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3499. doi:10.1158/1538-7445.AM2014-3499
Abstract 3536: Roles of miR-215 and regulatory mechanisms for its biogenesis in response to hypoxia in glioblastoma stem cells
Authors: Hu Jing , Sun Tao , Wang Hui , Wang Pingping , Fu Xiang-Dong , Li Qi-Jing , Wang Xiao-Fan
Published: May 2015

Glioblastoma (GBM) is the most common and aggressive primary brain tumor in adults, which is highly resistant to conventional therapies. In GBM, glioblastoma stem cells (GSCs) are critical for the tumor progression and resistance. The hypoxic tumor microenvironment has been reported to help maintain GSCs. However, the mechanism regulating responses of GSCs to hypoxia remains elusive. Abnormal microRNA processing is a contributing factor in various types of cancer including GBM. Using small RNA deep sequencing and Real-time PCR array, we found a group of miRNAs altered in the expression levels under hypoxia in the CD133+ GSCs. Further through a functional screen, we identified miR-215 as an important mediator for GSC growth and tumor progression in response to hypoxia. In addition, we also investigated the regulatory mechanisms for the biogenesis of the altered miRNAs under hypoxia. Interestingly, we found that hypoxia could regulate miRNA processing both transcriptionally and post-transcriptionally. These findings suggest that de-regulated miRNA biogenesis may have significant impacts on the activities of GSCs under hypoxia.

Citation Format: Jing Hu, Tao Sun, Hui Wang, Pingping Wang, Xiang-Dong Fu, Qi-Jing Li, Xiao-Fan Wang. Roles of miR-215 and regulatory mechanisms for its biogenesis in response to hypoxia in glioblastoma stem cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3536. doi:10.1158/1538-7445.AM2014-3536
Abstract 3558: The role of miRNA in PAX3-FKHR positive rhabdomyosarcoma
Authors: Muir Shannon , Nathanson Jason , Wilbert Melissa , Yeo Gene , Furnari Frank , Arden Karen , Cavenee Webster
Published: May 2015

Rhabdomyosarcoma is the most commonly diagnosed pediatric soft tissue cancer. Tumors may develop in several locations, but all tumors express markers of undifferentiated skeletal muscle tissue. Of the two main rhabdomyosarcoma subtypes, the alveolar subtype leads to a much poorer prognosis than the embryonal subtype. Alveolar rhabdomyosarcoma is characterized by a chromosomal translocation between either chromosomes 1 and 13, or more commonly 2 and 13, which creates the fusion genes PAX7-FKHR or PAX3-FKHR, respectively. The fusion genes encode a protein that contains the PAX DNA binding domain and the FKHR transactivation domain, but the mechanism of disease of the fusion protein has not yet been fully elucidated. Here we use gain of function and loss of function techniques to study the effect of PAX3-FKHR on miRNA expression. Using small RNA-seq, we show that PAX3-FKHR expression leads to an upregulation of microRNAs mir-495 and miR-543. Examination of the mir-495 and mir-543 genes revealed conserved PAX3 binding sites. We further investigate the effect of the microRNAs on rhabdomyosarcoma cell differentiation and tumorigenicity.

Citation Format: Shannon Muir, Jason Nathanson, Melissa Wilbert, Gene Yeo, Frank Furnari, Karen Arden, Webster Cavenee. The role of miRNA in PAX3-FKHR positive rhabdomyosarcoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3558. doi:10.1158/1538-7445.AM2014-3558
Abstract 3566: A robust and rapid targeted sequencing technology for iterative multiple genomic features in cancer
Authors: Lau Billy , Cushing Anna , Ji Hanlee
Published: May 2015

Targeted resequencing has enabled researchers and clinicians to analyze specific genomic regions of interest from cancer. For example, deep sequencing of cancer genes has allowed for the sensitive detection of rare somatic variants as well as oncogenic rearrangements that are otherwise difficult to observe with shotgun resequencing. Deep targeted resequencing of these regions can potentially provide prognostic information as well as guide treatment regimens. Current targeted resequencing methods, however, suffer from poor on-target performance, slow capture rates on the order of days, and significant manual effort. Existing commercial kits are extremely restricted for targeting custom regions of interest. Furthermore, libraries prepared for targeted resequencing purposes generally cannot be reused, thus limiting the number of replicates and follow-up experiments that can be performed for cancer samples.

We present APEX (attachment-based porous extension) capture, a platform that rapidly and robustly generates targeted DNA fragments for next-generation sequencing on the Illumina platform in as little as two hours. It is based on the principle that chemical reactions are rapidly enhanced in high surface-to-area ratio microenvironments when one reaction component is surface-bound. As a proof of concept, we covalently tethered next-generation sequencing compatible DNA libraries to a silica membrane found in conventional spin columns, performed target-specific primer extension, and eluted them for next-generation sequencing. Through serial dilutions of cytomegalovirus (CMV) and human DNA admixtures, we demonstrate CMV target enrichment down to single genome equivalents. In addition, using a tiling primer strategy we were able to robustly capture and resequence entire bacterial genomes.

Because our targeted resequencing technology preserves the original sample library, we can sequentially target different regions of interest in a series of guided experiments. We performed multi-tiered analyses of colon biopsies, where we deep resequence cancer genes as well as survey fro cancer-related pathogens such as bacterial genomes that are co-purified in the sample. APEX capture therefore enables researchers to aggressively and rapidly characterize cancer genomic material, where large-scale targeting panels are followed by precise discriminatory ones that can guide diagnosis and treatment.

Citation Format: Billy Lau, Anna Cushing, Hanlee Ji. A robust and rapid targeted sequencing technology for iterative multiple genomic features in cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3566. doi:10.1158/1538-7445.AM2014-3566
Abstract 3949: Microrna142 3p promotes tumorinitiating and radioresistant properties in malignant pediatric brain tumors
Authors: Chiou Shih-Hwa
Published: May 2015

Primary central nervous system (CNS) atypical teratoid/rhabdoid tumor (AT/RT) is an extremely malignant pediatric brain tumor in infancy and childhood. It has been reported that subpopulation of CD133+ cells isolated from ATRT tumors present cancer stem-like and radioresistant property. However, the exact biomolecular mechanism of ATRT or AT/RT-CD133+ is still unclear. In this study, we firstly showed that CD133-positive AT/RT cells (CD133+) have a pluripotent differentiation ability and the capability of malignant cells to be highly resistant to ionizing radiation (IR). Using microRNA array and quantitative RT-PCR, our data showed that expression of miR142-3p was lower in AT/RT-CD133+ than in AT/RT-CD133−. MiR142-3p overexpression significantly inhibited self-renewal and tumorigenicity of AT/RT-CD133+. On the contrary, silencing of endogenous miR142-3p dramatically increased tumor-initiating and stem-like cell capacities in ATRT or AT/RT-CD133− cells, and further promoted the mesenchymal-transitional and radioresistant properties of ATRT cells. Most importantly, therapeutic delivery of miR142-3p in ATRT effectively reduced its lethality by blocking tumor growth, repressing invasiveness, increasing radiosensitivity, and prolonging survival time in orthotropic-transplanted NOD-SCID mice. These results demonstrate the prospect of developing novel miRNA-based strategies to block the stem-like and radioresistant property of malignant pediatric brain cancer stem cells.

Citation Format: Shih-Hwa Chiou. Microrna142 3p promotes tumorinitiating and radioresistant properties in malignant pediatric brain tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3949. doi:10.1158/1538-7445.AM2014-3949