bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2050: Persistent STAT3 signaling contributes to the resistance of anti cancer drugs doxorubicin and cisplatin, and MEK inhibitor AZD6244 in human sarcoma cells
Authors: Wu Xiaojuan , Xiao Hui , Li Chenglong , Lin Jiayuh
Published: August 2015

Signal Transducer and Activator of Transcription 3 (STAT3) signaling pathway is activated in response to cytokines and growth factors. Persistently activated STAT3 are frequently detected in many types of human cancers and are believed to play an important role in their oncogenesis. Stat3 has been classified as a proto-oncogene because activated STAT3 can mediate oncogenic transformation in cultured cells and tumor formation in nude mice. We previously demonstrated that persistently activated STAT3 is frequently detected in human sarcoma such as rhabdomyosarcoma. To further examine the role of persistent TAT3 signaling plays in rhabdomyosarcoma, we used genetic approaches to either knock-down the expression of STAT3 using short hairpin RNA (ShRNA) or expressed constitutive active STAT3 protein. Knock-down the expression of STAT3 in rhabdomyosarcoma cells that express persistent STAT3 signaling, reduced the cell viability and sensitized cells to anti-cancer drugs doxorubicin and cisplatin. On the other hand, expression of constitutive active STAT3 protein in rhabdomyosarcoma cell lines lacking persistent STAT3 signaling decreased the sensitivity of those cells to doxorubicin and cisplatin. Furthermore, a MEK inhibitor, AZD6244 that is being tested in human cancer clinical trials unexpectedly induced STAT3 phosphorylation suggesting inducing of STAT3 may induce intrinsic resistance to MEK inhibitor. Accordingly, knock down of STAT3 sensitize sarcoma cells to MEK inhibitor treatments. To further translate our finding to potential cancer therapy, we tested a small molecular STAT3-selective inhibitor LY5 in sarcoma cells expressing persistent STAT3 signaling. Our data demonstrated that LY5 inhibited STAT3 phosphorylation, STAT3 DNA binding activity, and the migration of sarcoma cells. In addition, the combination of LY5 with anti-cancer drugs doxorubicin and cisplatin and the MEK inhibitor, AZD6244 also shown stronger inhibitory effects than single agent alone. In summary, our results using both genetic and small molecule approaches demonstrated that persistent STAT3 signaling contributes to the resistance of anti-cancer drugs doxorubicin and cisplatin, and MEK inhibitor in human sarcoma cells. They also implicated a potential cancer therapy strategy with the combination of STAT3 inhibitor with doxorubicin, cisplatin, and MEK inhibitor in sarcoma cells expressing persistent STAT3 signaling.

Citation Format: Xiaojuan Wu, Hui Xiao, Chenglong Li, Jiayuh Lin. Persistent STAT3 signaling contributes to the resistance of anti cancer drugs doxorubicin and cisplatin, and MEK inhibitor AZD6244 in human sarcoma cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2050. doi:10.1158/1538-7445.AM2015-2050
Abstract 2060: DUSP6 phosphatase as a tumor suppressor in non-small cell lung cancer
Authors: Perona Rosario , Moncho-Amor Veronica , Ibanez de Caceres Inmaculada , Pintado-Berninches Laura , Martin Ester , Quintanilla Miguel , Cortes-Sempere María , Sastre Leandro , Belda-Iniesta Cristobal
Published: August 2015

DUSP6/MKP3 a Map Kinase phosphatase involved in dephosphorylation and inactivation of ERK1/2 is a key gene whose expression in NSCLC is regulated by the DUSP1 Map Kinase phosphatase. Here we show that DUSP6 plays a major role in the regulation of cell migration, motility and tumor growth. We have found upregulation in the expression of several genes involved in Epidermal to mesenchimal transition in NSCLC-DUSP6 depleted cells. Furthermore ERK5 expression and phosphorylation, but not ERK1/2 is greatly enhanced in these cells. Finally the data obtained in RNAseq studies carried out in DUSP6 depleted cells identified several genes such as VAV3, RUNXR2, LEF1, FGFR2 whose expression is upregulated in these cells and affecting cellular functions such as integrin mediated cell adhesion and focal adhesion supporting the changes in cell adhesion and motility observed in DUSP6 depleted cells. TGFβ pathway is activated in these cells as we have observed phosphorylation of SMAD3 and consequently inhibition of TGFβ reduces the expression of EMT genes. Finally in silico studies from Gene Expression Omnibus and The Cancer Genome Atlas databases indicates that those NSCLC patients with low expression of DUSP6 are those with the worst prognosis.

Citation Format: Rosario Perona, Veronica Moncho-Amor, Inmaculada Ibanez de Caceres, Laura Pintado-Berninches, Ester Martin, Miguel Quintanilla, María Cortes-Sempere, Leandro Sastre, Cristobal Belda-Iniesta. DUSP6 phosphatase as a tumor suppressor in non-small cell lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2060. doi:10.1158/1538-7445.AM2015-2060
Abstract 2061: CHIP functions as tumor suppressor in prostate cancer through suppression of ROS-inflammasome in a chaperone-dependent mechanism
Authors: Said Neveen A. , Larner James
Published: August 2015

Prostate Cancer (PCa) is a disease closely associated with aging. Normal aging is driven by oxidative stress, resulting in accumulation of markers of protein and lipid/phospholipid damage with subsequent activation of inflammatory/oncogenic transcription factors NFκB and AP-1. Indeed, the tumor milieu characterized by ROS signaling, and NFκB- and AP1-activation is thought to be important in fueling cancer progression and metastasis. CHIP is a U-box-type E3-ubiquitin ligase that exerts a protective role against oxidative stress by targeting damaged proteins for proteasomal degradation to maintain tissue homeostasis. CHIP induces ubiquitylation and degradation of substrates, including oncogenes and oxidatively damaged, and misfolded proteins. We hypothesized that CHIP suppresses prostate tumor progression by inhibiting ROS generation and downstream oncogenic pathways. CHIP depletion in PCa cells significantly increased their anchorage-independent growth, migration and invasiveness. PCa cells depleted of CHIP exhibited increased ROS generation (H2O2) and expression of markers of oxidative stress 8-isoprostane, NOQ1, NNMT and sulfiredoxin with subsequent activation NFκB and AP-1. The increased activation of NFκB and AP-1 in CHIP-depleted cells was ROS-induced as it was inhibited by N-acetyl cysteine (NAC) or knockdown of NOQ1. Pharmacologic inhibition of Hsp90 inhibited basal and ROS-induced NFκB and AP-1 activation as well as their invasive phenotype suggesting that the effect of CHIP is in part chaperone-dependent. Furthermore, in subcutaneous and orthotopic xenografts, PCa cells depleted of CHIP exhibited significantly accelerated growth compared to controls. Molecular characterization of CHIP-depleted tumors revealed significantly increased proliferation, increased oxidative stress and inflammation with increased nuclear localization of NFκB and AP-1. Collectively, our data suggest a novel tumor suppressor role of CHIP in PCa mediated through inhibition of ROS-induced inflammation.

Citation Format: Neveen A. Said, James Larner. CHIP functions as tumor suppressor in prostate cancer through suppression of ROS-inflammasome in a chaperone-dependent mechanism. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2061. doi:10.1158/1538-7445.AM2015-2061
Abstract 2063: A novel ELAVL1-TYK2 fusion protein drives STAT3/5 activation and PIM-1 expression, survival and growth in the MOLM-16 acute myeloid leukemia cell line
Authors: McEachern Kristen , Keeton Erika , Dillman Keith , Ye Minwei , Stengel Chloe , Chen Huawei , Wang Suping , Grosskurth Shaun , Gale Rosemary E. , Linch David C. , Khwaja Asim , Lai Zhongwu , Huszar Dennis
Published: August 2015

Pim kinases are upregulated in leukemias and lymphomas and mediate cell growth and survival. Acute myeloid leukemia cell lines sensitive to the pan-Pim kinase inhibitor AZD1208 showed elevated pSTAT5 and Pim-1 expression that correlated with known tyrosine kinase mutations. However, one of the most sensitive lines, MOLM-16, lacked a reported driver mutation. RNA transcriptome sequencing identified a novel fusion gene comprised of the TYK2 kinase domain fused to the N-terminus of the RNA binding protein ELAVL1/HuR. The genes are co-localized within ∼2 MB on the short arm of chromosome 19, at position 13.2. The fusion results in loss of the Tyk2 pseudokinase domain, which negatively regulates kinase activity, suggesting a gain of function genetic alteration. Tyk2 was highly expressed and found to be amplified in MOLM-16 cells, with a higher degree of amplification of the exons encoding the kinase domain. Knockdown experiments demonstrated that down-regulation of the fusion gene, but not of the wild type ELAVL1 or TYK2 transcripts, resulted in suppression of STAT3/5 phosphorylation, Pim1 levels, and proliferation. Conversely, expression of the ELAVL1-TYK2 fusion protein in FDCP1 cells was shown to confer IL-3 independent growth. The fusion was not identified in screening of ∼200 AML patient samples or through analysis of TCGA data suggesting that it occurs at a low frequency, but interestingly NPM1-Tyk2 fusions have recently been described in cutaneous CD30-positive lymphoproliferative disorders (Velusamy et al. Blood 2014).

Citation Format: Kristen McEachern, Erika Keeton, Keith Dillman, Minwei Ye, Chloe Stengel, Huawei Chen, Suping Wang, Shaun Grosskurth, Rosemary E. Gale, David C. Linch, Asim Khwaja, Zhongwu Lai, Dennis Huszar. A novel ELAVL1-TYK2 fusion protein drives STAT3/5 activation and PIM-1 expression, survival and growth in the MOLM-16 acute myeloid leukemia cell line. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2063. doi:10.1158/1538-7445.AM2015-2063
Abstract 2071: Disruption of TGFβ-mediated tumor suppression in a Tpl2 model of cutaneous squamous cell carcinoma
Authors: Falkenberg Lauren , Gwilliam J. Curtis , Decicco-Skinner Katie L.
Published: August 2015

Skin cancer is the most common form of cancer in the United States, with an estimated two million cases diagnosed annually, and incidence continues to increase. Tpl2 (Map3k8) is a serine threonine protein kinase in the mitogen-activated protein kinase (MAPK) signal transduction cascade. Previous studies have shown that Tpl2-/- mice develop significantly more skin tumors than matched wildtype controls and keratinocytes harvested from Tpl2-/- mice display multiple biomarkers for cutaneous squamous cell carcinoma (cSCC.) We have previously established that crosstalk between Tpl2-/- keratinocytes and dermal fibroblasts contributes to the transformative potential of our model, and we hypothesized that one of the possible mechanisms driving this transformation was dysregulation of the TGFβ pathway, which plays multiple roles in carcinogenesis but is known to have an anti-proliferative function in noncancerous cells. Using various techniques, including real-time polymerase chain reaction, matrix-metalloproteinase zymography, in vitro angiogenesis assays, and immunoblotting, we saw that both Tpl2-/- keratinocytes and dermal fibroblasts secreted less TGFβ and displayed significant dysregulation of multiple components of the TGFβ pathway. In summary, our studies demonstrate that loss of Tpl2 results in disruption of TGFβ-mediated tumor suppression, and that disruption is in part caused by abnormal signalling from fibroblasts in the tumor microenvironment.

Citation Format: Lauren Falkenberg, J. Curtis Gwilliam, Katie L. Decicco-Skinner. Disruption of TGFβ-mediated tumor suppression in a Tpl2 model of cutaneous squamous cell carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2071. doi:10.1158/1538-7445.AM2015-2071
Abstract 2079: FGFR1/MAPK pathway regulates transcription factor brachyury-driven epithelial-mesenchymal transition in lung cancer
Authors: Hu Yunping , Hsu Wesley
Published: August 2015

Lung cancer is the leading cause of cancer-related death worldwide. Failure of treatment is attributed to recurrence, metastasis, and chemotherapeutic resistance. Previous studies have demonstrated that the transcription factor brachyury is a potential regulator of epithelial-mesenchymal transition (EMT) and plays a role in tumor resistance to conventional antitumor therapies. However, the mechanisms underlining brachyury-induced EMT are unknown. Recent studies have revealed that fibroblast growth factor (FGF)/ fibroblast growth factor receptor (FGFR) signaling is activated in lung cancer, and suppression of FGFR results in cell growth inhibition and apoptosis. Here we investigated the role of FGFR signaling, a critical component of mesoderm induction during normal embryogenesis and a regulator of normal brachyury expression, in brachyury-driven lung cancer. Quantitative RT-PCR and Western Blot analysis showed that the chemotherapy-insensitive/metastatic lung cancer cell line H460 and adenocarcinoma tissues in patients had significantly high expressions of brachyury and FGFR1, 3 and 4 compared to the chemotherapy-sensitive/non-metastatic lung cancer cell line H440 and normal tissues adjacent to tumor. To further explore the association between FGFR and brachyury expression and its function, we specifically silenced FGFR using siRNAs for FGFR1, 2, 3 and 4 in H460 cells or overexpressed FGFR1, 3 and 4 in H441 cells. We found that only FGFR1 inhibition led to suppression of brachyury, whereas overexpression of FGFR1 induced brachyury expression, suggesting that brachyury is a potential target of FGFR1. Immunofluorescence staining and RT-PCR showed that silencing of FGFR1 led to inhibitions of brachyury and EMT (an increased epithelial marker E-cadherin expression and decreased mesenchymal biomarker Snail expression) in H460 cells. Overexpression of FGFR1 promoted EMT in H441 cells, but this effect was suppressed by concomitant knockdown of brachyury. In addition, the mitogen-activated protein kinases (MAPK)/extracellular signal-related kinase (ERK) inhibitor, PD184352, suppressed FGF1-upregulated brachyury and Snail expressions and invasion in H460 cells. PD184352 also inhibited FGFR1 overexpression-induced brachyury expression, EMT and invasion in H440 cells, indicating an important regulatory role of FGFR1 in brachyury-driven EMT through MAPK/ERK. These findings provide novel insights into the molecular mechanism of brachyury-driven EMT and highlight the FGFR1/MAPK/ERK pathway as a new therapeutic target for brachyury-driven lung cancer.

Citation Format: Yunping Hu, Wesley Hsu. FGFR1/MAPK pathway regulates transcription factor brachyury-driven epithelial-mesenchymal transition in lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2079. doi:10.1158/1538-7445.AM2015-2079
Abstract 2083: TFAP4 inhibits differentiation of MYCN-amplified neuroblastoma
Authors: Zhang Shuobo , Lopez Gonzalo , Yu Jiyang , Silva Jose , Kadenhe-Chiweshe Angela , Califano Andrea , Yamashiro Darrell
Published: August 2015

Background. Despite the identification of MYCN-amplification as an adverse prognostic marker in neuroblastoma, no drugs that target MYCN have yet been developed. An alternative approach is to identify genes synthetically lethal to MYCN-amplified neuroblastoma. Through a whole genome shRNA screen, we have previously identified transcription factor AP4 (TFAP4) as a synthetic lethal gene of MYCN-amplified neuroblastoma. Silencing of TFAP4, both in vitro and in vivo inhibited the growth of MYCN-amplified neuroblastoma. TFAP4 has previously reported to repress neuronal specific genes in non-neuronal cells (Kim et al, 2006). Here, we investigate the function of TFAP4 in inhibiting differentiation of MYCN-amplified neuroblastoma.

Methods: Master regulators specifically associated to MYCN amplified patients were identified by MARINA (Master Regulator Inference algorithm). TFAP4 expression was measured by real-time PCR, Western blot and immunohistochemistry. GAP43 expression was detected by Western blot. Gene expression changes after silencing of TFAP4 or retinoic acid treatment was determined by RNAseq.

Results: TFAP4 was identified as a master regulator specifically in MYCN amplified neuroblastoma patients in both NCI-TARGET dataset and a published GEO dataset (Kocak et al, 2013). We determined that TFAP4 is a direct target of MYCN and TFAP4 expression was highly correlated with Stage 4, MYCN-amplified neuroblastoma in both patient datasets. Neuronal markers TUBB3 and GAP43 expression were inversely correlated with TFAP4 expression in TARGET (P = 9.77e-03 and P = 7.78e-08). Silencing TFAP4 in MYCN amplified cells induced a differentiated phenotype, as shown by markedly increased the number of neurites, and an increase in the neuronal differentiation marker GAP43. Forty hours after silencing TFAP4, we observed 415 genes that were significantly up-regulated and 457 genes down-regulated (P<0.01). Genes with increased expression were enriched for gene ontology terms associated with neuronal activity. Gene signature of TFAP4 also showed a strong correlation with the treatment signature of the differentiation agent retinoic acid in SKNDZ.

Conclusions. We demonstrated that TFAP4 is an important downstream effector of MYCN, that inhibits the neuronal differentiatin of MYCN-amplified neuroblastoma. This may provide novel targets for the treatment of neuroblastoma.

Citation Format: Shuobo Zhang, Gonzalo Lopez, Jiyang Yu, Jose Silva, Angela Kadenhe-Chiweshe, Andrea Califano, Darrell Yamashiro. TFAP4 inhibits differentiation of MYCN-amplified neuroblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2083. doi:10.1158/1538-7445.AM2015-2083
Abstract 2087: Regulation of p53 function by KLF5
Authors: Yang Yizeng , Zhou Taicheng , Zong Zhen , Lomberk Gwen , Urrutia Raul , Katz Jonathan
Published: August 2015

Background: The transcription factor Krüppel-like factor 5 (KLF5) is essential for normal homeostasis, stemness, and tumorigenesis. Typically, KLF5 functions as a transcriptional activator, including in esophageal epithelia where KLF5 promotes keratinocyte proliferation and migration by activating EGFR and ILK, respectively, and prevents transformation by activating Notch1. Yet, the function of KLF5 is context dependent, and mutation of the tumor suppressor p53 switches KLF5 from a transcriptional activator to a repressor of p21Waf1/Cip1 and from pro-proliferative to anti-proliferative. Interestingly, KLF5 also inhibits p53 itself, though the mechanism of this inhibition is unknown. We hypothesized that the functional switch of KLF5 from transcriptional activator to repressor involves the recruitment of a co-repressor.

Methods: Primary human esophageal keratinocytes immortalized with hTERT were stably infected with inducible lentivirus containing shRNA against KLF5 or Sin3A. p53 mRNA and protein levels were assessed by real-time quantitative PCR and Western blotting, respectively, and p53 protein stability was evaluated by pulse-chase experiments. We employed ChIP across a 4 kb region on the p53 gene, from 2 kb upstream to 2 kb downstream of the transcription start site, to identify the region of KLF5 binding on p53. Promoter activity was assessed with a series of luciferase reporters containing varying lengths of p53 and its upstream regulatory region. Protein-protein interaction was examined by co-IP.

Results: KLF5 knockdown significantly increased p53 both at the mRNA and protein levels. In unstressed cells p53 protein undergoes rapid degradation such that p53 levels are typically low. Yet, with KLF5 knockdown, despite increased p53 protein levels, we saw no changes in p53 protein stability. By ChIP, we identified a region of KLF5 binding to p53 between -600 to -356 bp upstream of the transcription start site. Several other KLF family members can interact with the co-repressor Sin3A, and we found that Sin3A also complexed with KLF5 in human primary esophageal keratinocytes. Knockdown of Sin3A abolished the inhibitory effect of KLF5 on p53, resulting in elevated p53 expression, growth inhibition, and increased apoptosis.

Conclusion: KLF5 transcriptionally represses p53 via the co-repressor Sin3A. Thus, KLF5 promotes cell proliferation in normal keratinocytes by both activating pro-growth signals and inhibiting the brake machinery of the cell cycle.

Citation Format: Yizeng Yang, Taicheng Zhou, Zhen Zong, Gwen Lomberk, Raul Urrutia, Jonathan Katz. Regulation of p53 function by KLF5. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2087. doi:10.1158/1538-7445.AM2015-2087
Abstract 2089: Role of Cdx2 in maintainining differentiated chromatin structure during tumor progression
Authors: Perekatt Ansu O. , Rana Ritu , Kumar Namit , Valdez Michael , Verzi Michael
Published: August 2015

Tumor progression often coincides with the appearance of tumor cells with dedifferentiated or stem-like properties; the ontogeny of these cells is unclear, but they are thought to carry a more malignant capacity than their differentiated counterparts. Cdx2 is a transcriptional factor that specifies cell fate of the intestinal epithelium. Epigenetic studies have demonstrated that Cdx2 loss alters chromatin structure in the intestinal epithelium. Cdx2 loss is often observed during colon tumor progression and is correlated with de-differentiation. Our current study is aimed to test the hypothesis that the Cdx2 prevents tumor progression by maintaining the tumor epigenome. To this end, we have generated two mouse models to determine the effect of Cdx2 loss on chromatin structure in a tumorigenic environment and in primary tumors. Our current studies are aimed at comparing Cdx2 binding sites on the genome in a normal and tumorigenic environment, and in defining how Cdx2 loss affects chromatin structure in primary tumors. These studies will shed light on how alterations in chromatin structure can contribute to the epigenomic mechanisms of colon tumor progression.

Note: This abstract was not presented at the meeting.

Citation Format: Ansu O. Perekatt, Ritu Rana, Namit Kumar, Michael Valdez, Michael Verzi. Role of Cdx2 in maintainining differentiated chromatin structure during tumor progression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2089. doi:10.1158/1538-7445.AM2015-2089
Abstract 2105: An EWS-FLI1/MRTF gene regulatory network in Ewing sarcoma
Authors: Katschnig Anna M. , Kauer Maximilian , Schwentner Raphaela , Aryee Dave N.T , Lawlor Elizabeth , Kovar Heinrich
Published: August 2015

The pediatric bone cancer Ewing sarcoma (ES) is characterized by the expression of the chimeric transcription factor EWS-FLI1, derived from a chromosomal translocation t(11;22)(q24;q12). The early onset of metastasis in ES leads to poor overall survival and limited treatment options. Remodeling of the actin cytoskeleton is a prerequisite of a tumor cell in order to become metastatic. Previous studies suggest that EWS-FLI1 alters cell morphology and deregulates various cytoskeletal genes. The major point of convergence of extracellular signals influencing cytoskeletal regulation is the Rho pathway. Activation of Rho GTPases promotes polymerization of monomeric G-actin into stress fibers (F-actin) thereby releasing the myocardin-related transcription factor family (MRTF-A, MRTF-B) of transcriptional coactivators. MRTFs translocate to the nucleus and interact with the transcription factor serum response factor (SRF) bound on CArG promoter elements of genes. However, the ternary complex factor (TCF) family, a class of transcriptional coactivators activated downstream of Ras signaling, competes with MRTFs for SRF binding in presence of ets binding sites adjacent to the CArG consensus sequence. The ets-transcription factor EWS-FLI1 has been shown to substitute for TCFs and interact with SRF. We hypothesized that EWS-FLI1 competes with MRTFs for SRF binding and uncouples target gene expression from Rho signaling. In combinatorial knockdown experiments of MRTFA/B and EWS-FLI1 we demonstrate that global changes in gene expression elicited by EWS-FLI1 silencing largely depend on the presence of MRTFs, suggesting that the fusion oncogene prohibits MRTF activity. Using gene expression analysis we identified six different patterns of MRTF/EWS-FLI1 interaction, with predominance of EWS-FLI1 inversely regulating MRTF target genes. Furthermore, we found MRTF transcriptional effects are already present in the absence of serum in A673 ES cells. MRTFB, however, is cytoplasmic under serum starved conditions and translocates to the nucleus only upon serum stimulation unless latrunculin B (LatB), an actin polymerization inhibitor, is added. In contrast, MRTFA is constitutively nuclear even under serum starved conditions or upon LatB treatment indicating that MRTFA might be uncoupled from actin regulation. These findings reveal a previously unknown role of MRTFs and the “CArGome” in EWS-FLI1 driven transcriptional dysregulation. Supported by the Liddy Shriver Sarcoma Initiative.

Citation Format: Anna M. Katschnig, Maximilian Kauer, Raphaela Schwentner, Dave N.T Aryee, Elizabeth Lawlor, Heinrich Kovar. An EWS-FLI1/MRTF gene regulatory network in Ewing sarcoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2105. doi:10.1158/1538-7445.AM2015-2105
Abstract 2108: Regulation of nucleotide metabolism by mutant p53 contributes to its gain-of-function activities
Authors: Martinez Luis A. , Kollareddy Madhusudhan
Published: August 2015

Mutant p53 (mtp53) is an oncogene that drives cancer cell proliferation. We found that mtp53 associates with the promoters of numerous nucleotide metabolism genes (NMG). Mtp53 knockdown reduced NMG expression and substantially depleted nucleotide pools. Decreased GTP levels correlated with reduced activation of GTP dependent proteins (GTPases) and reduced invasiveness. Addition of exogenous GTP restored the invasiveness of mtp53 knockdown cells, suggesting that mtp53 promotes invasion by increasing GTP. Additionally, mtp53 creates a dependency on the nucleotide salvage pathway enzyme deoxycytidine kinase (dCK) for the maintenance of a proper balance in dNTP pools required for proliferation. These data indicate that mtp53 harboring cells have acquired a synthetic sick or lethal phenotype relationship with the nucleotide salvage pathway. Finally, elevated expression of NMG correlates with mutant p53 status and poor prognosis in breast cancer patients. Thus, mtp53's control of nucleotide biosynthesis has both a driving and sustaining role in cancer development.

Citation Format: Luis A. Martinez, Madhusudhan Kollareddy. Regulation of nucleotide metabolism by mutant p53 contributes to its gain-of-function activities. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2108. doi:10.1158/1538-7445.AM2015-2108
Abstract 2109: Short isoform ARID5B protein is expressed in endometrial cancer cell lines by usage of the second translation initiation codon
Authors: Kawamata Norihiko , Itakuara Keiichi
Published: August 2015

ARID5B, also known as MRF2, is a transcription factor with the ARID-type DNA binding domain. The ARID5B gene generates two types of mRNA isoforms (short and long isoforms) using alternative first exons; the long and short mRNA isoforms encode 130kDa long and 100kDa short isoform ARID5B proteins, respectively. The ARID5B gene is highly mutated in endometrial cancer. Nonsense and frame-shift mutations, generating premature termination codons, are common. This transcription factor may play a role as a tumor suppressor in development of endometrial cancer and the inactivation of the gene by the mutations may contribute to carcinogenesis of this cancer.

To characterize functional significance of the gene products in this cancer, we analyzed the ARID5B gene in three endometrial cancer cell lines (Ishikawa, HEC59, and KLE).

Two cell lines, Ishikawa and HEC59, predominantly expressed the long isoform mRNA while KLE expressed both the long and short isoform mRNAs. However, surprisingly, all the cell lines predominantly expressed the 100 kDa protein, which was detected by an antibody recognizing the ARID-domain. To characterize the 100kDa protein, we generated rabbit anti-serum recognizing amino-terminus of the long isoform protein, which failed to detect the 100kDa protein. The data suggested that the 100kDa protein lacked the N-terminus of the long isoform ARID5B protein. Internal ribosome entry site (IRES) activity was found in the sequence between the first and second ATGs of the long isoform ARID5B mRNA by the bicistronic reporter gene assay. Our data suggested that the second translation initiation codon was used to generate the short isoform ARID5B protein in the cell lines.

To elucidate functional property of this short isoform ARID5B protein expressed in the cell lines, we suppressed the expression of the ARID5B protein by shRNA. The expression was decreased by 50% using the shRNA constructs. Colony formation assay of Ishikawa cells transfected with the shRNA constructs showed that repression of ARID5B short isoform protein decreased the number of the colonies. In summary, the short isoform ARID5B protein generated by usage of the second translation initiation site was indispensable in these endometrial cancer cells.

Citation Format: Norihiko Kawamata, Keiichi Itakuara. Short isoform ARID5B protein is expressed in endometrial cancer cell lines by usage of the second translation initiation codon. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2109. doi:10.1158/1538-7445.AM2015-2109
Abstract 211: A novel FRET assay for the intracellular activation of ADC linkers
Authors: Lee Byoung-Chul , Chalouni Cecile , Nalle Sam , Doll Sophia , Darwish Martine , Mellman Ira , Vandlen Richard
Published: August 2015

Evaluating the intracellular activation of ADC by a novel FRET assay.

Despite the recent success of ADCs as cancer therapeutics, their mechanisms of action are not fully understood. In this study, we developed ADCs using a novel fluorescence resonance energy transfer (FRET) linker in order to facilitate monitoring the details of intracellular uptake, vesicular trafficking and payload release. In the FRET linker, the cathepsin-cleavable dipeptide of val-cit was inserted between a fluorescence donor Alexa Fluor 488 (or later, fluorescein) and an acceptor tetramethylrhodamine (TAMRA). Upon cleavage of the val-cit linker, a fluorescence signal from Alexa Fluor 488 or fluorescein is expected to appear as a probe for monitoring intracellular activation of ADC.

We used two in-vitro human cancer cell lines, SKBR3 and PC3. SKBR3 is a Her2-positive breast cancer cell line and PC3 is a prostate cancer cell line that has been engineered to express a prostate cancer-specific growth factor receptor, tomoregulin-2 (TenB2). We assessed the intracellular processing of the FRET conjugates using flow cytometry. We found that 74% and 41% of the linker was cleaved in PC3 and SKBR3 cells after 20 hours of treatment, respectively.

In the live-celling imaging of the FRET probe in SKBR3 and PC3 cells, Anti-Her2 and TenB2 FRET ADCs showed a remarkable difference of internalization pathways and cleavage following receptor-mediated endocytosis. In PC3 cells, FRET ADCs were internalized and congregated into a certain localized area during the early stage of uptake in 2 to 3 hours. The ADC-containing vesicles were spread over the cytosol and near the plasma membrane in a subset of cells. A similar directed localization at the early stage of uptake was not observed for the SKBR3 cells. The endosomal vesicles in SKBR3s were spread over the cytosol during the course of ADC internalization. The amount of the release payload in the cytosol of SKBR3s was 3-fold greater than that of PC3 cells, suggesting that the endosomal/lysosomal membrane of SKBR3 may be more permeable than that of PC3.

In summary, our ADC FRET probes allowed for the facile, robust and non-invasive evaluation of intracellular processing for ADC linkers. Quantitative measurements for the rate of antigen-mediated intracellular cleavage of the FRET linker as well as cytosolic release could be evaluated by our FRET probes.

Citation Format: Byoung-Chul Lee, Cecile Chalouni, Sam Nalle, Sophia Doll, Martine Darwish, Ira Mellman, Richard Vandlen. A novel FRET assay for the intracellular activation of ADC linkers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 211. doi:10.1158/1538-7445.AM2015-211
Abstract 2122: RNA editing reveals new regulatory functions in tumorigenesis
Authors: Hu Landian
Published: August 2015

RNA Editing is a widespread post-transcriptional event that could increase the diversity of transcriptome. In humans, the most common type of RNA editing is adenosine-to-inosine (A-to-I) editing which is catalyzed by a family of enzymes called adenosine deaminase acting on RNA (ADARs). Over 90% A-to-I RNA editing sites locate in Alu elements, the most abundant transposable elements in human genome. We picked up 83 CEU individuals and 89 YRI individuals for RNA editing analysis. 94,315 credible sites are obtained after series of strict computational procedures. Those genes whose transcripts harbor numerous RNA sites are found to be enriched in several cancer-related pathways such as small cell lung cancer pathway, p53 signaling pathway and apoptosis pathway. Our work provides new insight into the regulation mechanism of RNA editing acting on tumorigenesis.

Citation Format: Landian Hu. RNA editing reveals new regulatory functions in tumorigenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2122. doi:10.1158/1538-7445.AM2015-2122
Abstract 213: Evaluating high risk BI-RADS 4 mammographic lesions: a pilot trial of textural analysis (TA) as a supplement to digital mammography (DM)
Authors: Gordon Melissa R. , Borazanci Erkut , Maki Daniel D. , Korn Ron L.
Published: August 2015

Background: Although mammographic lesions classified as BI-RADS 4 are considered suspicious for carcinoma - by definition - and biopsy (bx) is therefore always recommended, only 20-30% of such lesions ultimately yield malignancy at biopsy. TA is an ancillary non-subjective quantitative analysis platform that can be performed as an add-on to routine mammographic analysis. The ability of TA to characterize lesions with high rates of heterogeneity led us to hypothesize that TA, as a supplement to routine DM analysis, might aid in selecting patients (pts) for bx vs. serial observation.

Methods: A retrospective analysis was performed on 40 screening pts with no history of cancer, with newly identified mammographic densities characterized as BI-RADS 4. Lesions measured 1-3 cm size and had no associated microcalcifications. Twenty pts had confirmed malignant biopsy while the other 20 pts served as age matched controls with similar lesion characteristics but ultimate benign biopsy result. For each lesion, TA was performed on a 1×1cm region of interest (ROI), along with a secondary control ROI of normal breast tissue.

Results: Pts with malignant and benign bx had a median age of 53 years (range 28-83) vs. 52 years (range 32-75) (p = NS) and median nodule size 1.5 cm (range 1.0-2.9 cm) vs. 1.3 cm (range 1.0-2.5 cm) (p = NS), respectively. No differences between the groups were seen in menopausal status nor parenchymal density. The mean positive pixel (MPP) value was most predictive of malignancies at a Spatial Scale Frequency (SSF) filter level of 0.8. The median MPP for malignant lesions was 577 vs 730 for benign lesions (p = 0.033). There was no difference between MPP or other TA parameters in normal tissue of either group.

Conclusion: TA, as a supplement to DM, may help stratify which women with high-risk BI-RADS 4 densities that better benefit from biopsy vs. those that might be safely followed with serial surveillance. Larger scale studies will be needed to validate these findings and are currently ongoing.

Citation Format: Melissa R. Gordon, Erkut Borazanci, Daniel D. Maki, Ron L. Korn. Evaluating high risk BI-RADS 4 mammographic lesions: a pilot trial of textural analysis (TA) as a supplement to digital mammography (DM). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 213. doi:10.1158/1538-7445.AM2015-213
Abstract 2136: Correlating the expression of protein kinase C isozymes with the transformed phenotype in colorectal cancer
Authors: Dowling Catríona M. , Phelan James J. , Cathcart Mary Clare , Mehigan Brian , McCormick Paul , Dalton Tara , Coffey John C. , O'Sullivan Jacintha , Kiely Patrick A.
Published: August 2015

Protein Kinase C (PKC) is a family of serine/threonine kinases that are involved in almost every signal transduction cascade in cells. PKC enzymes are activated by second messangers and respond to specific environmental cues. The development of PKC modulators with anti-cancer therapeutic potential is a major target in cancer. However, this task is made difficult because PKC has an important role to play in normal processes and the PKC family consists of at least 9 different isozymes. Our hypothesis is that PKC expression, activity and localisation are altered as colon epithelial cells switch from normal to the transformed state. We are confident that being able to recognise these changes has the potential to be used as a biomarker and prognostic marker in the early detection of colon cancer.

Real Time PCR was utilised to compare the gene expression profile of 9 PKC isozymes in matched normal and cancer tissue samples. We found differential expression of the conventional PKCs, with PRKCB (the gene coding for PKC Beta II) being significantly downregulated in the cancer tissue of over 90% of patients (p<0.01,n = 22). This change was closely linked to the progression of the disease. Using tissue microarrays, we also demonstrated significant alterations in both epithelial and stromal levels of PKC Beta II between the patients normal and diseased tissue. The level of PKC Beta II in the diseased tissue was significantly decreased across patients (p<0.01,n = 164) and we associate the decreased expression with tumour stage. In conclusion we suggest that differential expression of PKC Beta II may be an important mechanism regulating the change to the transformed phenotype in colon cancer.

Citation Format: Catríona M. Dowling, James J. Phelan, Mary Clare Cathcart, Brian Mehigan, Paul McCormick, Tara Dalton, John C. Coffey, Jacintha O'Sullivan, Patrick A. Kiely. Correlating the expression of protein kinase C isozymes with the transformed phenotype in colorectal cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2136. doi:10.1158/1538-7445.AM2015-2136
Abstract 2137: The effect of the transcription factor MAZ on kRAS transcription: a role for the G-quadruplex
Authors: Batra Harshul , Brooks Tracy A.
Published: August 2015

kRAS is a GTPase protein affecting normal cell proliferation, division, and apoptosis. It is mutated in >30% of all cancers, particularly in 60-90% of pancreatic cancers. Mutated kRAS leads to continuous activity and uncontrolled proliferation. To date there have been no successful clinical agents targeting mutant kRAS activity. Modulating kRAS expression has shown anti-proliferative promise as an approach in pancreatic cancer models, but is not a main avenue of pursuit for clinical development due to lack of a molecular target. Our lab is focused on the regulation of kRAS transcription through a variety of G-rich regions of DNA in the promoter capable of forming non-B-DNA structures termed G-quadruplexes (G4s). In the current study, we sought to determine the effect of the transcription factor MAZ on the regulation of kRAS, with a particular focus on the three putative G4-forming regions (herein termed near, mid, and far in reference to their relative proximity to the transcriptional start site). In a study of the kRAS promoter using a series of luciferase plasmids, MAZ expression led to a concentration-dependent decrease in promoter activity. This action was localized to interactions with the mid-G4-forming region. In the context of a more multifaceted intracellular mileu as found in the pancreatic cancer cell lines MiaPaCa-2, Panc-1, and BxPc3, the effects of MAZ were much more complex. In particular, 48 hr post-transfection with MAZ expression plasmids, monitoring of kRAS transcription revealed a cell line-specific modulation with an increase in kRAS expression in Panc-1 cells only, confirming a previous study, and no changes noted in either the MiaPaCa-2 or the Bx-Pc3 cells. Further studies are ongoing evaluating the direct binding of MAZ to the various G-rich regions in those cell lines, as well as determining the DNA structure (single-stranded, double-stranded, or G4-DNA) to which the MAZ protein is binding. Ultimately, an understanding of the regulation of this G-rich region of DNA, by MAZ or any other transcription factors such as Sp1 or p53, is an important part of the larger puzzle leading to a targeted drug discovery program focused on G4-regulation. Ultimately, G4-stabilization-mediated down regulation of kRAS has high potential for anti-cancer efficacy in pancreatic cancers, where there is a dire need for novel therapeutic development.

Citation Format: Harshul Batra, Tracy A. Brooks. The effect of the transcription factor MAZ on kRAS transcription: a role for the G-quadruplex. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2137. doi:10.1158/1538-7445.AM2015-2137
Abstract 2143: A 3D system to elucidate a direct role for epiregulin mistrafficking in epithelial transformation
Authors: Singh Bhuminder , Bogatcheva Galina , Coffey Robert J.
Published: August 2015

The EGF receptor (EGFR) ligand epiregulin (EREG) is delivered preferentially to the basolateral cell surface of polarized MDCK cells. Previously, 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 an EREG mutation (R147stop) in human tumors that we now report mistrafficks to the apical surface. To test the hypothesis that apical mistrafficking of EREG can be a driver event, we have generated MDCK cells stably expressing inducible (Tet-ON) wild-type and Y156A mutant EREG. In 3D Matrigel cultures, we show that uninduced clones form normal-appearing cysts, regardless of EREG status. Induction of WT or mutant EREG leads to increased cyst diameter. However, only (Y156A)EREG-expressing cells form abnormal cysts with ectopic lumens and inward growth, features of in vivo transformation. Furthermore, we show that ligand processing and EGFR activity is required for EREG mistrafficking-induced ectopic lumen formation as preincubation with a broad-spectrum metalloprotease inhibitor, BB-94, abrogates ectopic lumen formation. We have extended these studies to polarizing human colon cancer cells, HCA-7, which preserve the trafficking fidelity of WT and mutant EREG. Furthermore, using EREG cytoplasmic domain as a bait in a yeast-2-hybrid screen, we have identified APOBEC3C as an interacting protein. APOBECs are a family of cytidine deaminases that have been identified as a major contributor to human cancer mutations. We have subsequently confirmed EREG-APOBEC3C interaction by co-immunoprecipitation. Results from MDCK cells stably expressing differentially inducible EREG (Tet-ON) and APOBEC3C (Cumate-ON) will be presented.

Note: This abstract was not presented at the meeting.

Citation Format: Bhuminder Singh, Galina Bogatcheva, Robert J. Coffey. A 3D system to elucidate a direct role for epiregulin mistrafficking in epithelial transformation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2143. doi:10.1158/1538-7445.AM2015-2143