bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3080: MYC antagonizes imatinib-induced differentiation in chronic myeloid leukemia cells through downregulation of p27KIP1
Authors: Leon Javier , Gomez-Casares M. Teresa , Ferrandiz Nuria , Lopez Carmen E. , Garcia-Alegria Eva , Albajar Marta , Vaque Jose P , Bretones Gabriel , Delgado M. Dolores
Published: August 2012

Imatinib is a BCR-ABL kinase inhibitor and the most used drug in chronic myeloid leukemia (CML). MYC (c-Myc) is a transcription factor frequently deregulated in human cancer that exerts multiple biological activities, including differentiation inhibition. We found that MYC expression is higher in bone marrow cells form CML patients at diagnosis, as compared with healthy controls. Moreover, high MYC levels at diagnosis correlated with a poorer response to imatinib treatment. One of the major characteristics of CML progression is the loss of cell differentiation. We showed that low concentrations of imatinib induce erythroid differentiation in the CML-derived K562 cell line. We have studied the effect of MYC on the differentiation induced by imatinib using K562 sublines carrying inducible (by zinc cation) or activable (by 4-hydoxy-tamoxifen) MYC alleles. In both cell lines, MYC largely prevented the erythroid differentiation induced by imatinib. The differentiation inhibition mediated by MYC is not due to increased proliferation of MYC-expressing cells or enhanced apoptosis of differentiated cells in the presence of MYC. We previously reported that p27/Kip1 (p27) overexpression induces erythroid differentiation in K562. Thus, we explored the effect of imatinib on p27 levels and we found that imatinib up-regulated p27 in a time- and concentration-dependent manner. siRNA-mediated silencing of p27 antagonized the erythroid differentiation induced by imatinib, indicating that p27 up-regulation is at least in part responsible for differentiation. MYC abrogated the imatinib-induced up-regulation of p27KIP1 concomitantly with the differentiation inhibition, suggesting that MYC inhibits imatinib-mediated differentiation of K562 cells by antagonizing p27 up-regulation. We previously reported that MYC induced SKP2, a component of the ubiquitin ligase complex that targets p27 for degradation. MYC was able to induce SKP2 in the presence of imatinib. Thus, MYC-mediated inhibition of the differentiating effect of imatinib in CML cells is in part a consequence of the SKP2 induction of MYC, which in turn would down-regulate p27. The results suggest that, although MYC deregulation does not directly confer resistance to imatinib, it might be a factor that contributes to progression of CML through the inhibition of differentiation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3080. doi:1538-7445.AM2012-3080
Abstract 3081: Crosstalk between Runx2 regulatory network and PI3K/Akt signaling pathway in breast cancer cells
Authors: Tandon Manish , Chen Zujian , Pratap Jitesh
Published: August 2012

The Runt-related transcription factor Runx2 is essential for skeletal development but is also aberrantly expressed in several cancers (e.g., breast, prostate and osteosarcoma) where it promotes cell migration, invasive and growth properties of cancer cells. The phosphatidylinositol 3′ kinase (PI3K)/Akt signaling pathway is linked to Runx2-dependent skeletogenesis, however, the mechanisms of Runx2 role in PI3K-Akt signaling in breast cancers, and its implication in cancer progression remains under investigated. To mechanistically determine the function of Runx2 in epidermal growth factor (EGF)-induced PI3K/Akt signaling and concomitantly ensuing downstream signaling events, we modulated Runx2 expression levels in immortalized normal MCF-10A and invasive MDA-MB-231 cells via lentiviral vector-mediated gene delivery. The Runx2 over-expression in EGF-stimulated MCF-10A cells increased the cellular response towards PI3K inhibitor LY294002 as indicated by a decline in Akt (Serine 473) phosphorylation (pAkt) levels. This effect could be restored by Runx2 suppression indicating the specificity of Runx2 function in Akt regulation. In MDA-MB-231 cells, Runx2 over-expression and, surprisingly, suppression profoundly inhibited pAkt suggesting that a threshold level of Runx2 is required for phosphorylation of Akt. The Runx2-mediated decline in pAkt was completely reversed by the Runx2-DNA binding mutant over-expression indicating that Runx2 DNA binding domain is essential for Akt inhibition. To further dissect the mechanism of Runx2-mediated regulation of pAkt levels in MDA-MB-231 cells, we examined the protein levels of phosphatases and kinases associated with PI3K/Akt signaling pathway. These analyses revealed that PH domain and leucine rich repeat protein phosphatase 1 (PHLPP1) levels were directly responsive to Runx2. Chromatin immunoprecipitation studies showed Runx2 recruitment on the proximal promoter of PHLPP1 suggesting that Runx2 mediates its effect on Akt activity, in part by regulating PHLPP1 expression. Furthermore, Runx2 overexpression increased nuclear translocation of a forkhead box transcription factor FoxO1, a downstream molecule of PI3K/Akt signaling indicating an indirect Runx2 affect on FoxO1 transcriptional activity in MDA-MB-231 cells as examined by western blot analysis and immunofluorescence studies. Together, our results identified clinically relevant and novel mechanisms of Runx2 regulatory network in PI3K/Akt signaling pathway that could have important consequences in targeting breast cancer-associated cell survival and invasive properties.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3081. doi:1538-7445.AM2012-3081
Abstract 3085: ERα phosphorylation on Ser167 in the development of endometrial cancer
Authors: Gonzalez Sandra Herrero , Cristofano Antonio Di
Published: August 2012

Endometrial carcinoma (EC) is the most common malignancy of the female genital tract. Type I (endometrioid) EC is associated with elevated estrogen signaling. One of the earliest genetic events in human type I EC is loss of the tumor suppressor gene PTEN. Loss of PTEN allows unrestrained activation of the AKT kinase, which, in turn, regulates such key processes as proliferation, survival, cell size, and mRNA translation. We have previously shown, using a Pten+/− mouse model, that the neoplastic lesions develop even in the absence of estrogen, suggesting that there might exist a functional and physiological link between loss of Pten and hormone-independent increased estrogen receptor activity. Previous studies have suggested that AKT can phosphorylate and activate estrogen receptor alpha (ERα) at Ser167, promoting the activation of this nuclear receptor both in vivo and in vitro, even in the absence of ligand. In order to define in vivo the role of ERα phosphorylation on Ser167 in normal endometrial physiology and during neoplastic transformation, we have designed a knock-in approach to introduce the S167A mutation in the mouse germline (ERαA/A). These mice are viable and fertile. We bred ERαA/A mice to Pten+/− mice to test whether ERα phosphorylation on Ser167 is required for the development of endometrial hyperplasia and carcinoma. We found that, at the age of 6 months, the uterus weight of ERαA/A/Pten+/− mice was significantly reduced compared to that of Pten+/− mice. To elucidate whether inhibition of ERα phosphorylation on Ser167 affects cell cycle progression, we analyzed BrdU incorporation in primary cultures of control and ERαA/A endometrial epithelial cells, and found that impairment of ERα phosphorylation on Ser167 leads to a significant reduction of the number of actively proliferating cells. In order to understand the molecular mechanisms responsible for this cell cycle impairment, we analyzed by real-time PCR the expression levels of cell cycle-related genes in the uterus of control and ERαA/A mice. Surprisingly, our results indicate that many genes related with the G2/M phase of the cell cycle, like Ccna2 or Ccnb1, and spindle formation, like Plk1 or Tpx2, are deregulated in ERαA/A uteri, compared to wild-type controls. In conclusion, our results indicate that ERα phosphorylation on Ser167 is an obligatory pathway for the development of endometrial lesions consequent to loss of Pten in endometrial cancer, at least in part by affecting the expression of cell cycle genes.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3085. doi:1538-7445.AM2012-3085
Abstract 3087: CD24 regulates tumor cell behaviour in a c-Src dependent fashion
Authors: Bretz Niko P. , Schlange Thomas , Salnikov Alexei V. , Noske Aurelia , Perne Claudia , Moldenhauer Gerhard , Kristiansen Glen , Altevogt Peter
Published: August 2012

CD24 is a glycosyl phosphatidylinositol (GPI)-anchored membrane protein that is frequently over-expressed in a variety of human carcinomas and is correlated with poor prognosis. In cancer cell lines, changes of CD24 expression can alter several cellular properties like invasion, proliferation and adhesion in vitro and tumor growth in vivo. However, little is known about the mechanisms by which CD24 mediates these effects. Here we have analyzed functional consequences of CD24 knock-down or over-expression in human cancer cell lines of different tumor entities (ovarian, lung, glioma). We show that presence or absence of CD24 was crucial to regulate the amount and activity of c-Src in lipid rafts. CD24-mediated effects on tyrosine-phosphorylated c-Src had impact on regulation of target genes affecting tumor cell invasion, proliferation and apoptosis. Invasive potential of tumor cells was controlled by the expression of the tissue factor pathway inhibitor-2 (TFPI-2), a potent inhibitor of extracellular matrix degradation that can block tumor cell invasion and metastasis. Silencing of CD24 or c-Src enhanced TFPI-2 expression and diminished invasion of SKOV3ip and A549 cells into matrigelTM. Conversely, over-expression of CD24 reduced TFPI-2 expression and enhanced invasion. Furthermore, an inverse correlation between expression of CD24 and TFPI-2 was observed by immunohistochemical analysis of primary breast cancers (N = 1174). TFPI-2 expression was highest in CD24 negative samples and lowered with increasing CD24 expression. Patients with a CD24 low/TFPI-2 high phenotype showed significantly better survival compared to CD24 high/TFPI-2 low patients (p=0.001). Additionally, STAT3 expression and phosphorylation were reduced by CD24 silencing. Diminished STAT3 activity was confirmed by specific reporter assays. Src depletion abrogated levels of p-STAT3 (Y705). In line with that, expression of classical STAT3 target genes like MCL-1, Cyclin D1 and Survivin was reduced after CD24 knock-down. Suppressed cell proliferation and enhanced apoptosis were observed in CD24 knock-down cells, suggesting a pivotal role of regulated target genes. An antibody to CD24 was effective in reducing tumor growth of lung- and pancreatic cancer xenografts in mice. Antibody treatment affected the level of Src-activity in the tumor and altered expression of STAT3 target genes. Our results provide evidence that CD24 regulates TFPI-2 and STAT3 activity via the c-Src non receptor tyrosin kinase. Targeting of CD24 by antibodies could represent a novel strategy for anti tumor therapy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3087. doi:1538-7445.AM2012-3087
Abstract 3091: A sumoylation-dependent transcriptional subprogram is required for Myc-driven tumorigenesis
Authors: Kessler Jessica D. , Kahle Kristopher T. , Sun Tingting , Meerbrey Kristen L. , Schlabach Michael R. , Schmitt Earlene M. , Skinner Samuel O. , Xu Qikai , Li Mamie Z. , Hartman Zachary C. , Rao Mitchell , Yu Peng , Dominguez-Vidana Rocio , Liang Anthony C. , Solimini Nicole L. , Bernardi Ronald J. , Yu Bing , Hsu Tiffany , Golding Ido , Luo Ji , Osborne C. Kent , Creighton Chad , Hilsenbeck Susan G. , Schiff Rachel , Shaw Chad A. , Elledge Stephen J. , Westbrook Thomas F.
Published: August 2012

Myc is an oncogenic transcription factor frequently dysregulated in human malignancies. While the transcriptional programs and other functions of Myc have been intensively studied, there remains no effective strategy for inhibiting Myc in patients. To search for pathways that support the Myc oncogenic program, we employed a next-generation RNAi screen for Myc-synthetic lethal (MySL) genes. Using this strategy, we have identified several cellular processes required to tolerate oncogenic Myc. Key among these is the core sumoylation machinery, and we define the Sumo-activating enzyme (SAE) as a central component in this MySL network. Loss of SAE drives synthetic lethality with Myc, and the enzymatic activity of SAE is required to support the Myc oncogenic state. Inactivation of SAE leads to mitotic catastrophe and cell death selectively upon Myc hyper-activation. Mechanistically, depletion of SAE switches a subprogram of Myc transcriptional targets governing mitotic spindle function from activated to repressed, a subprogram we term Sumoylation-dependent Myc Switchers, or SMS genes. Notably, SMS genes are required to tolerate Myc hyper-activation, and SAE and the SMS program are required for Myc-dependent breast cancer cell survival in vitro and tumor growth and progression in vivo. Importantly, patient survival significantly correlates with levels of SAE and SMS gene expression in Myc-high tumors. Collectively, these studies reveal a mitotic vulnerability of Myc-driven cancers and demonstrate that inhibiting sumoylation can selectively impair mitosis and survival in an oncogenic Myc-dependent manner. We propose that drugs targeting SAE and its downstream SMS targets may have therapeutic benefits for patients with Myc-driven cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3091. doi:1538-7445.AM2012-3091
Abstract 3093: Elucidation of c-KIT- dependent signaling to MITF
Authors: Phung Bengt , Sun Jianmin , Schepsky Alexander , Steingrimsson Eirikur , Rönnstrand Lars
Published: August 2012

MITF, the master regulator of melanocytes, is a basic basic- helix- loop-helix- leucine zipper transcription factor that is essential for the development of melanocytes. MITF is also a lineage- survival oncogene in melanoma and recently, it has been shown that germline mutations in MITF predispose to this malignant disease. In melanoma, MITF has been proposed to act as a rheostat where high levels of MITF contribute to a proliferative phenotype whereas low levels result in a more invasive phenotype. The biological output generated by high or low levels of MITF is well characterized. However, how the activity of MITF is regulated to mediate these effects is less well known. It has been proposed that MITF is phosphorylated upon ligand induced activation of the receptor tyrosine kinase c- KIT. C-KIT engages the MAP kinase signaling cascade where ERK2 directly phosphorylates Ser73 of MITF and P90RSK phosphorylates the Ser409 residue of MITF. As a consequence, MITF activity increases and the protein is subjected to ubiquitin dependent degradation.Here, we show for the first time that c-KIT induces phosphorylation of MITF through the survival PI3 kinase- AKT pathway, the P38 stress pathway as well as the well-known MAP kinase-ERK signaling pathway. In addition, we show that the SRC kinase upstream of ERK is essential for c-KIT induced MITF activation. Moreover, we used several c-KIT phosphorylation site mutants to map the specific sites that are involved in the mediating transcription activation potential of MITF. These mutants also show less effects on MITF-mediated proliferation.We have generated phosphorylation specific antibodies against Ser73 and Ser409 of MITF and characterized the phosphorylation pattern of MITF using these reagents. Our results suggest that there is a particular relationship between Ser73 and Ser409 with subsequent effects on function. Based on our findings, we propose that phosphorylation of MITF might act as negative feedback mechanism that keeps MITF activity in check. Our model challenges the current knowledge of MITF modulation and provides insights that may have a profound impact on our understanding of the role of MITF in melanocyte and melanoma biology.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3093. doi:1538-7445.AM2012-3093
Abstract 3098: ERCC1 gene copy number variations in resected non-small cell lung cancer
Authors: Friboulet Luc , Olaussen Ken A. , Valent Alexander , Tang Ximing , Tang Tao , Li Yuanyuan , Behrens Carmen , Vielh Philippe , Robin Angélique , Goubar Aïcha , Validire Pierre , Wistuba Ignacio , Soria Jean-Charles
Published: August 2012

Introduction: ERCC1 allows removal of branched DNA structures at essential steps in DNA repair pathways such as Nucleotide Excision Repair (NER), Fanconia-dependent Interstrand Cross-Link Repair (ICL-R), long-patch base excision repair (BER), and Single Strand Annealing (SSA). ERCC1 expression is a prognostic and predictive biomarker of chemotherapy effect in NSCLC. The most frequent method to determine ERCC1 status is the evaluation of gene expression (IHC or RT-PCR). Here, we studied the prevalence of ERCC1 copy number variation (CNV) using FISH. Methods: FISH was performed with Poseidon™ dual color probe set, ERCC1 (19q13) and ZNF443 (19p13) on a panel of 17 NSCLC cell lines and 324 FFPE tumor samples from resected NSCLC patients (103 from IGR, Villejuif, France, 221 from MDACC, Houston, USA). Gene amplification (GA) and high polysomy (HP) were defined following actual consensus rules for EGFR assessment. Results: ERCC1 copy number was increased (>2) in a large majority of cell lines (13/17) of which three displayed gene amplification (GA) (H2342, CN=5; H1299, CN>12; HOP62, CN>15). In human samples, overall ERCC1 gene abnormalities were detected in 65 cases (20.1%) including 31 GA (6.9%, the majority with clusters) and 34 HP (13.7%, more frequent in squamous cell carcinomas). In patients with ERCC1 expression status available (immunohistochemistry) we found a non-significant trend for higher ERCC1 expression in HP patients compared to GA and low CNV patients (P=0.06). Despite a 3-fold increase in IC50 of cisplatin in cell lines with ERCC1 gene amplification compared to normal cells (with no CNV) the ERCC1 FISH status seemed unable to predict chemosensitivity in resected NSCLC patients. In patients not treated with chemotherapy, DFS was significantly longer in patients with normal ERCC1 FISH status of the tumor compared to patients with ERCC1 gene amplification (P=0.02). Conclusion: ERCC1 copy number variation is a frequent event in NSCLC and could reflect genomic instability, but the clinical relevance of this observation needs further investigation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3098. doi:1538-7445.AM2012-3098
Abstract 31: Activated phosphatidylinositol-3 kinase: An oncogene that increases gap junctional, intercellular communication
Authors: Geletu Mulu , Greer Samantha , Raptis Leda
Published: August 2012

Gap junctions are channels that connect the cytoplasm of adjacent cells. Gap junctional, intercellular communication (GJIC) is blocked in cells transformed by oncogenes such as the middle Tumor antigen of polyoma virus (mT), an oncoprotein which associates with and is tyrosine-phosphorylated by cSrc family members. Specific phosphotyrosines provide docking sites for the phosphotyrosine binding domain of Shc (mT-tyr250) and the SH2 domain of the phosphatidylinositol 3-kinase (mT-tyr315), resulting in the activation of their downstream signaling cascades, Ras/Raf/Erk and PI-3 kinase/Akt, respectively. To examine the effect of these mT-initiated pathways upon gap junctional communication and neoplasia, GJIC was assessed in mT-mutant-expressing, rat liver epithelial T51B cells which normally have extensive GJIC, using a novel technique of in situ electroporation we developed. The results show that although low levels of wt-mT are sufficient to interrupt gap junctional communication, GJIC suppression still requires an intact tyr250 site, that is activation of the Ras pathway. In sharp contrast, activation of the PI-3 kinase pathway is not required for GJIC suppression. It is remarkable that T51B cells expressing a mutant deficient in binding of Shc, hence activation of the Ras pathway, are still morphologically transformed and do grow into tumors in syngeneic rats, albeit with an altered morphology. These results indicate that GJIC suppression requires Ras but not PI-3 kinase/Akt signalling, and is independent of full neoplastic conversion in rat liver epithelial cells. Since PI3k activation by mT increases GJIC, we examined whether PI3k might have a positive role upon GJIC. The results showed that PI3k pharmacological inhibition eliminates GJIC, concomitant with apoptosis induction. We next expressed a form of PI3k rendered constitutively active through the addition of a myristylation signal (myr-PI3k). The results demonstrated that myr-PI3k causes an increase in GJIC. Therefore PI3k, although in an activated form it can act as an oncogene, it plays a positive role upon gap junctional, intercellular communication.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 31. doi:1538-7445.AM2012-31
Abstract 310: Oncogene-specific regulation of immune responses during tumorigenesis
Authors: Juric Vladislava , Wang Chunmei , Chen Xin , Bishop J. Michael
Published: August 2012

Oncogenes can induce inflammatory signaling, leading to the recruitment of immune cells to tumor site. Immune cells can further influence tumor development by either restraining or facilitating tumorigenesis. Here, we investigated how distinct oncogenes shape the immune responses during tumorigenesis in vivo. We utilized several established mouse models of liver cancer in which tumorigenesis is driven by the overexpression of MYC, MET, or AKT+RAS oncogenes. We evaluated the immune cells at early vs. late stages of tumor development by immunohistochemical staining of liver tissues using lineage-specific immune cell markers. In addition, we assessed activation of the pro-inflammatory NFκB pathway by immunoblot detection of IkBα and phospho-Ser(536)-NFκB levels in liver lysates, as well as by immunohistochemical analysis of NFκB (p65) nuclear localization. Histological analysis revealed leukocytic clustering around hepatocytes in pre-neoplastic livers in MYC and AKT+RAS, but not in the MET liver tumor model. Moreover, the leukocyte profiles identified in pre-neoplastic livers and liver tumors were different in MYC vs. AKT+RAS model. Finally, we found that the NFκB pathway was inactive in MYC and AKT+RAS tumors, but hyperactivated in MET tumors. In summary, our results suggest that the type of tumor-initiating oncogenic lesion profoundly influences the identity of recruited immune cells and activation of NFκB pathway in the course of tumorigenesis. We are currently in the process of elucidating the mechanisms behind oncogene-mediated regulation of immune responses, which may reveal novel targets for anti-cancer therapy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 310. doi:1538-7445.AM2012-310
Abstract 3100: Identifying novel chromosomal fusions out of RNAseq data from oropharyngeal cancers
Authors: Wang Vivian W. , Laborde Rebecca R. , Asmann Yan W. , Li Yang , Ma Jian , Eckloff Bruce W. , Tombers Nicole M. , Olsen Steven M. , Moore Eric J. , Olsen Kerry D. , Smith David I.
Published: August 2012

Chromosome rearrangements that result in gene fusions have important roles in the initial steps of tumorigenesis especially in leukemias and lymphomas, but the biological and clinical impact of gene fusions in the common solid tumor has been less appreciated. We previously generated RNAseq data from 12 oropharyngeal tumors along with matched normal tissue from the same patients. We utilized and compared two bioinformatic pipelines, SnowShoes-FTD and FusionHunter to identify two novel fusion events involving of MGST3-ZMAT5 and MS4A7-DKFZp586P0123. These fusions were validated utilizing. Reverse transcriptase polymerase chain reaction (RT-PCR) and Sanger sequencing validated. In the first translocation the 5′ UTR of MGST3 was fused with exon 1 of ZMAT5, and in the second translocation the exon 2 of MS4A7 was fused with 3′ UTR of DKFZp586P0123. These precise fusions were not present in the matched normal tissue, however, they were only found in the single tumor in which they were identified. Given a growing and powerful set of high-throughput massively parallel sequencing technologies, the future for genome-wide analysis of chromosome rearrangement appears open in many directions. Ultimately, we hope that this integrative methodology will elucidate key aspects of tumor biology as well as generate novel targets for cancer diagnostics and therapy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3100. doi:1538-7445.AM2012-3100
Abstract 3103: Differences in the DDR enzymes activation kinetics between normal and cancer cells could be utilized to achieve targeted cellular sensitivity towards genotoxic agents
Authors: Khalil Hilal S. , Tummala Hemanth , Hupp Tedd , Zhelev Nikolai
Published: August 2012

The DNA damage response (DDR) pathway is a complex signalling network that is activated when eukaryotic cells undergo DNA damage caused by exposure to genotoxic agents. Depending upon the type and scale of DNA damage, the DDR can either cause cell cycle arrest if the DNA lesions are repairable, or trigger apoptosis if the DNA lesions are beyond the repair capacity of the cell. A variety of enzymes take part in this complex signalling network that decides cellular fate after DNA damage, and is comprised of kinases, phosphatases and glycosylases. Here we explored the kinetics of activation of these key enzymes, the rates of their product formation and the interplay between them that impact the decision making process of the cell. We exposed normal (MCF10A, HaCat) and breast cancer cell lines (MCF7, MDA MB231, MDA MB468) to different combinations of radiomimetic drugs, UV radiation and enzyme inhibitors in a time and dose dependent manner and looked at the resulting enzyme activation rates of DDR kinases such as ATM, ATR, Chk1, Chk2, and DDR phosphatases PP2A and WIP1. We studied the effects of such enzymatic activation on their substrates P53, E2F1, BRCA1 and γ-H2AX and examined its outcome on cell survival. Firstly, we have identified key differences in enzyme activation kinetics that impacted the cell fate decision between normal and cancer cell lines. In addition we established treatment regimes based on those differences that bias the cell fate decision towards apoptosis in cancer cells and cell cycle arrest and repair of DNA in their normal counterparts. Thus, we have demonstrated a link between the rate of induction of different enzyme activities in the DDR pathway, the resulting product formation and cellular sensitivity to genotoxic agents. We have successfully used such information to devise a combinatorial drug treatment regime that is selective for cancer cells. Elucidation of the enzyme activation kinetics in a time and dose dependent manner of the proteins governing cell fate decision can be used to achieve targeted cellular selectivity and sensitivity with lesser side effects on normal cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3103. doi:1538-7445.AM2012-3103
Abstract 3108: Exploring base excision repair variants as potential defining events in cancer patient populations and characterization of models for pre-clinical studies
Authors: Riches Lucy C. , Brown Helen , Dalgliesh Gillian , Fletcher Lynn , Sherwood James , Shea Kerry , Lau Alan , O'Connor Mark J.
Published: August 2012

Background Deficiencies in base excision repair (BER) may increase the cell's dependence on alternative repair pathways and so define a patient population for novel inhibitors of DNA damage repair. Notably, cancer associated molecular events in DNA polymerase beta (POLα) have been described in various disease backgrounds including colorectal cancer (CRC), gastric, breast and prostate (1-5). Although functional consequence has been linked to certain POLα mutations and splice variants, their prevalence in defined disease segments remains largely undefined. Aim We set out to ascertain the prevalence of POLα deficiencies in CRC, and subsequently investigated protein and gene expression profiling as alternative methods for defining BER defects in cancer. Methods/Results A comprehensive study of clinical (CRC; N=64), tumour explant (CRC; N=36) and cell line (mixed origin; N=280) samples revealed that cancer-associated mutations in the polymerase encoding domain of POLα (1-3) are not common events in CRC. Additionally, a previously described splice variant of POLα lacking part of the polymerase domain (3-4), was assayed and found to be expressed at similar levels in CRC and normal tissue. This would suggest that expression of the POLα splice variant is not a defining element of CRC in western populations. Expression profiling using Fluidigm Taqman revealed a subset of BER genes more highly expressed in CRC clinical samples and mixed cell lines compared with normal tissue. While protein expression profiles did not reflect relative gene expression in selected cell line models, we observed an additional low molecular weight band in a subset of cells profiled for POLB expression. Furthermore, we report variations in the expression of the scaffold protein XRCC1 in colorectal and breast cell lines and propose that this could also provide a mechanism for variation in DNA repair capacity. Summary Although from our studies sequencing and transcript analysis of POLα do not provide suitable markers of BER deficiency in CRC, we report aberrant banding patterns in POLα by Western blotting and variation in XRCC1 protein expression in a subset of cell lines analyzed. We hypothesize that these variants may confer a BER deficient phenotype. 1. Lang, T. et al. (2004Proc. Natl Acad. Sci. USA, 101, 6074-6079. 2. Tan, Xiao-Hui, et al Cancer Letters: 2005: 220: 101-114 3. Wang, L et al Cancer Research, 1992; 52:4824-4827 4. Bhattacharyya, N. et al. (1997) Proc. Natl Acad. Sci. USA, 94, 10324-10329

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3108. doi:1538-7445.AM2012-3108
Abstract 3113: Effects of an up-regulated ATR-CHEK1 pathway in head and neck squamous cell carcinomas
Authors: Parikh Rahul A. , Lewis Dale W. , Gollin Susanne M.
Published: August 2012

Background: The ATR-CHEK1 pathway is upregulated in a subset of head and neck squamous cell carcinoma (HNSCC) cell lines with loss of the G1 cell cycle checkpoint. We hypothesized that the upregulated ATR-CHEK1 pathway protects HNSCC from mitotic catastrophe (cell death) by enhancing the G2M phase checkpoint and that inhibition of this pathway may sensitize HNSCC to DNA damaging agents. Methods and Results: In HNSCC, we observed gain, amplification or translocation of the ATR gene in 8 of 20 cell lines studied; whereas the CHEK1 gene is partially lost in 12 of 20 cell lines. ATR and CHEK1 FISH in paraffin sections of primary head and neck squamous cell carcinomas confirmed our findings in HNSCC cell lines. We observed that both ATR and CHEK1 are overexpressed by quantitative PCR in five of eight HNSCC cell lines with loss of the G1 cell cycle checkpoint. Inhibition of ATR and CHEK1 with caffeine or specific inhibition with the respective siRNAs results in increased susceptibility of HNSCC to ionizing radiation by clonogenic cell survival assay. siRNA-mediated ATR or CHEK1 knockdown leads to loss of G2M checkpoint accumulation and increased sub-G0 apoptotic population by flow cytometric analysis. The mechanism of cell death after ATR and CHEK1 knockdown is by mitotic catastrophe, as evidenced by increased premature chromatin condensation. Conclusions: The ATR-CHEK1 pathway is up-regulated in a subset of HNSCC with loss of G1 phase cell cycle checkpoint. The up-regulated ATR-CHEK1 pathway protects these HNSCC from mitotic catastrophe by enhancing the G2M phase cell cycle checkpoints and inhibition of ATR and/or CHEK1 increase the sensitivity of the HNSCC to ionizing radiation. We conclude that inhibition of the up-regulated ATR-CHEK1 pathway in HNSCC may potentiate or increase the efficacy of the current therapeutic modalities for HNSCC.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3113. doi:1538-7445.AM2012-3113
Abstract 3178: Can DNA from archived formalin-fixed paraffin embedded (FFPE) cancer tissues be used for somatic mutation analysis in next generation sequencing
Authors: Palescandolo Emanuele , Jones Robert , Raza Alina , Sunkavalli Ashwini , Brastianos Priscilla K. , Ducar Matthew , Go Christina , Roden Christine , Hatton Charles , Hanna Megan , Mills Adri , Adusumilli Ravali , Kumar Prateek , Meyerson Matthew L. , William Hahn C. , MacConaill Laura , Hummelen Paul Van
Published: August 2012

Formalin-fixed paraffin embedded (FFPE) tissues are far more abundant in most tissue banks and pathology departments than fresh or fresh frozen (FF) samples, but typically yield varying degrees of degraded DNA as a result of the fixation process. The quality of FFPE tissues may vary based on a number of factors such as: age of the block, fixation time, storage and handling conditions. Being able to harness the power of next generation sequencing technologies to genomically characterize these abundant and diverse achieved samples would be tremendously valuable to the cancer research community and would enable the use of this material for clinical purposes. The goal of this study was to assess the performance of FFPE samples in next generation sequencing applications. We monitored close to 100 samples from tumor and normal tissues of FFPE, FF and blood origin. These samples underwent several processes for whole-exome or targeted sequencing, including DNA fragmentation, size selection, library preparation, and hybrid-capture enrichment. We observed variable performance across these samples at several of the above steps, which correlated predominantly with FFPE tissues and the age of block. Other quality metrics that showed relative lower performance of FFPE DNA were the cluster density, duplication rate or library complexity. Most FFPE samples still generated good quality sequence, however, older FFPE blocks, over 10 years, may need more input DNA or higher sequence depth to reach minimum coverage if somatic mutation analysis is the goal. There was no difference in performance between DNA from tumor and normal tissues.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3178. doi:1538-7445.AM2012-3178
Abstract 3180: A fast solution to NGS library preparation with low nanogram DNA input
Authors: Liu Pingfang , Lohman Greg , Cantor Eric , Langhorst Brad , Apone Lynne , Munafo Daniela , Sumner Christine , Stewart Fiona , Evans Thomas C. , Dimalanta Eileen T. , Davis Theodore
Published: August 2012

Next generation sequencing (NGS) has significantly impacted cancer genetics, enabling a comprehensive characterization of genomic abnormalities in the cancer genome at very high resolution. By delivering massive DNA sequences at unprecedented speed and cost, NGS promises to make personalized genome-based diagnosis and targeted cancer therapy a reality in the foreseeable future. To date, library construction with clinical samples has been a challenge, primarily due to the limited quantities of sample DNA available. To overcome this challenge, we have developed a fast library preparation method using novel NEBNext reagents and adaptors. This method enables library construction from a minimal quantity of DNA (< 5 ng), and can be used for both intact and fragmented DNA. Moreover, the workflow is compatible with multiple NGS platforms.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3180. doi:1538-7445.AM2012-3180
Abstract 3185: Rapid and efficient methods for preparing rRNA-depleted and directional RNA-Seq libraries from low-input and FFPE RNA samples
Authors: Sooknanan Roy , Hitchen John
Published: August 2012

Massively parallel sequencing of cDNA libraries (RNA-Seq) is rapidly becoming the preferred method for transcript profiling, and analysis of novel transcripts, novel isoforms, alternative splice sites, rare transcripts and cSNPs, compared to microarrays. Preparing NGS RNA-Seq libraries typically require first isolating rRNA-depleted RNA or enriching for poly(A)+ mRNA. However, total RNA isolated from formalin-fixed, paraffin-embedded (FFPE) patient tissue samples is normally fragmented, making it not suitable for rRNA-depletion using some commercially available kits or poly(A)+ enrichment, which results in a 3′ sequence bias. Here, we present an efficient “single-pass” rRNA-depletion method (Ribo-Zero™ technology) for use with as little as 100 ng total RNA input from either intact or fragmented (e.g., FFPE) RNA samples. Additionally, an improved, more user-friendly version of the ScriptSeq™ RNA-Seq method (ScriptSeq™ v2) is used to rapidly prepare directional (∼99% strandedness) RNA-Seq libraries in about 2.5 hours, in a single-tube workflow, from either the intact or fragmented Ribo-Zero™ treated RNA samples. The ScriptSeq™ method does not require end-polishing, adaptor-ligation, cDNA fragmentation, or gel-size selection. The combined Ribo-Zero™ and ScriptSeq™ workflow is completed in about 5 hours, generating cluster-ready NGS libraries that contain <2 % of reads that map to rRNA sequences (nuclear- and mitochondrial-encoded) and ∼98% of reads that map to the genome, while maintaining the representation of coding and non-coding transcripts, independent of polyadenylation. This reduction in rRNA sequence reads improves sequence depth and coverage of mRNA, and increases the percentage of uniquely mapped reads required for transcriptome analysis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3185. doi:1538-7445.AM2012-3185
Abstract 3186: Comparative analysis of different total RNA sequencing approaches
Authors: Sumner Christine J. , Munafo Daniela , McReynolds Larry , Langhorst Brad , Liu Ping , Apone Lynne , Stewart Fiona , Dimalanta Eileen , Davis Theodore
Published: August 2012

Initial transcriptomic studies largely relied on hybridization-based microarray technologies and offered a limited ability to fully catalogue and quantify the diverse RNA molecules that are expressed from genomes over wide ranges of levels. Massively parallel cDNA sequencing, or Total RNA-Seq, has allowed many advances in the characterization and quantification of transcriptomes; an offer a new appreciation for the complexity of the Transcriptome, encompassing a multitude of previously unknown coding and non-coding RNA species, particularly small RNAs, including micro RNAs. This work investigates the performance of different strategies for Total RNA-Seq using multiple next generation sequencing platforms. Standard RNA-sequencing approaches generally require double-stranded cDNA Synthesis, which erases RNA strand information. Synthesis of a randomly primed double-stranded cDNA followed by addition of adaptors for next-generation sequencing leads to the loss of information about which strand was present in the original mRNA template. The polarity of the transcript is important for correct annotation of novel genes, identification of antisense transcripts with potential regulatory roles, and for correct determination of gene expression levels in the presence of antisense transcripts. Here, we examine the performance of strand-specific RNA libraries made by direct ligation of adaptor on to the RNA. We analyze the effect of different RNA fragmentation methods (divalent cations plus heat versus enzymatic fragmentation) and we provide a comparative data analysis (library complexity, continuity of gene coverage, strand specificity and 3′and 5′-end bias analysis). Identification and analysis of small RNA by deep sequencing requires preparation of a di-tagged cDNA library, which leads to adaptor-dimer formation that strongly contaminates the library. We have developed a novel method to generate di-tagged small RNA libraries free of adapter-dimer contamination without introducing any additional enzymatic steps or gel purifications. This method has optimized the 3′adaptor-ligation reaction to recover and to increase representation of the 2′-O-modified RNAs present in a biological sample. To reduce cost and increase sample throughput we have developed a barcode strategy to tag samples during library construction. The multiplexed libraries can then be pooled together before size selection, reducing the number of steps in the workflow. This technique reduces bias by ligation, increases representation of modified small RNAs and simplifies workflow during library construction for small RNA analysis and discovery.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3186. doi:1538-7445.AM2012-3186
Abstract 3196: In vivo drug sensitivity testing of CTCs isolated using the FMSA device
Authors: Gallant Jean-Nicolas , Cheng Hairong , Harouaka Ramdane , Zheng Siyang , El-Deiry Wafik S.
Published: August 2012

BACKGROUND: Cells disseminated from primary epithelial tumors into peripheral blood, called circulating tumor cells (CTCs), can be monitored to assess metastases and to provide a surrogate marker of treatment response; indeed, the enumeration of fixed CTCs with the Johnson and Johnson Veridex CellSearch System is FDA-approved for assessment of patient prognosis in metastatic breast, prostate, and colorectal cancer. The opportunity to isolate viable CTCs using the Flexible Micro Spring Array (FMSA) device – a novel microfluidic device that enriches CTCs by two physical parameters: size and flexibility – offers the possibility of testing cancer treatments in real time and rapidly providing information back to clinicians regarding the best course of treatment. OBJECTIVES: In this study, we evaluated the possibility of isolating viable CTCs from human blood using the FMSA device, growing these cells in a mouse xenograft model, and determining successful treatments for metastatic disease. METHODS: GFP-labeled HCT-116 cells were spiked into samples of blood from healthy donors to mimic the blood of patients with metastatic disease. This spiked blood was filtered using the FMSA device, and the recovered cells were implanted into the flanks of immunodeficient mice for study. A series of in vivo experiments were carried out to investigate the effect of chemotherapy on the resulting tumors. These tumors were molecularly characterized, compared to tumors derived from non-filtered cells, and analyzed for potential differences resulting from FMSA enrichment. Studies are ongoing to further investigate the use of this model and approach to culture CTCs from patients. RESULTS & CONCLUSIONS: Cancer cells isolated from blood using the FMSA can proliferate when implanted subcutaneously into the flanks of immunodeficient mice – developing into tumors within two weeks. Although they grow at rates that depend on the number of cancer cells found in each mL of blood, these tumors provide a means for testing the effects of chemotherapy on CTCs in vivo. Using this model, treatments targeted towards CTCs could be assessed and provide real-time information to clinicians regarding treatment interventions for patients with metastatic cancer and detectable CTCs. It may also be possible to use this model as the primary mode for CTC culture as the in vivo environment may provide soluble factors, cell-to-cell interactions, and physical parameters that are not easily mimicked by in vitro culture. The approach has potential to help with efforts to develop personalized therapy options for patients with advanced cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3196. doi:1538-7445.AM2012-3196