bluuebunny/crossref_metadata_2025_split huggingface dataset:
Abstract 1853: Single-cell multiplexed profiling of protein-level changes induced by EGFR inhibitor gefitinib
Authors: Holcomb Ilona , Dakshinamoorthy Gajalakshmi , Liu Benjamin , Unger Marc , Ramakrishnan Ramesh , Gong Haibiao 
Published: July 2016

Abstract
Single-cell molecular analysis has become increasingly important in biomedical research, especially in the field of cancer research, where cell heterogeneity is a characteristic feature. We developed a multiplexed single-cell protein detection assay for the C1™ system that can isolate and process 96 individual cells in parallel. The assay uses oligonucleotide-conjugated antibodies, which produce a detectable DNA amplicon only when they bind to the target protein. The resulting DNA amplicons are quantified by qPCR on the Biomark™ HD system. Data are analyzed using the Singular™ Analysis Toolset. A total of 50 antibody binders were developed for this assay to target proteins related to apoptosis, cell cycle, cell proliferation, tumor suppressors, biomarkers, stem cells, growth factors and proteins associated with specific cancers, such as breast and prostate cancers. The assay can be performed in a 48-plex format, which yields over 4,600 datapoints in two days. Assay sensitivity and specificity were assessed using recombinant proteins and various cell lysates. To demonstrate the usefulness of this assay, we used it to profile the protein level changes in A431 cells treated with epidermal growth factor receptor (EGFR) inhibitor gefitinib. EGFR is a member of ErbB family of type I tyrosine kinases, whose overexpression and mutations are involved in a variety of cancers. While much effort has been made to investigate the consequences of EGFR activation or inhibition, hardly any information is available regarding how EGFR regulation changes protein expression in single cells. Using a multiplexed protein assay, we discovered some interesting co-expression patterns of proteins in single cells. We also demonstrated that TNFRSF10B was upregulated, while MET, GATA3, BRCA1, CCNB1, Ki-67 and CCNA2 were downregulated by gefitinib treatment. Notably, the reduction of the cell cycle-related proteins CCNB1 and CCNA2 and the cell proliferation marker Ki-67 was consistent with the inhibited cell growth by gefitinib. In addition, we measured mRNA expression to investigate the association between protein and mRNA levels in single cells. Single-cell analysis—determining protein changes and analyzing the correlation between different proteins at the single-cell level—yields valuable information that is not achievable using bulk-cell analysis.
Citation Format: Ilona Holcomb, Gajalakshmi Dakshinamoorthy, Benjamin Liu, Marc Unger, Ramesh Ramakrishnan, Haibiao Gong. Single-cell multiplexed profiling of protein-level changes induced by EGFR inhibitor gefitinib. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1853.
Abstract 1860: MDM2 stabilizes and induces HIF-1α levels during reoxygenation of cancer cells
Authors: Kanagasabai Thanigaivelan , Chand Rohin , Kaur AmyAman , Dhandayuthapani Sivanesan , Bracho Olena , Rathinavelu Appu 
Published: July 2016

Abstract
Hypoxia stimulates several pathways that are critical to cancer cell growth and survival, including activation of vascular endothelial growth factor (VEGF) transcription. Overexpression of VEGF and the extent of neo-angiogenesis are closely correlated with increase in tumor size and cancer metastases. The main regulator of hypoxia-induced angiogenesis is the hypoxia inducible factor (HIF)-1α which regulates the transcription of various target genes including VEGF. Stabilization of HIF-1α in cancer cells could enable tumor progression and results in poor survival. In this respect, the multifunctional mouse double minute 2 homolog (MDM2) oncoprotein has been gaining significant amount of attention. The MDM2 oncoprotein has been shown to exert both p53-dependent and p53-independent roles in oncogenesis. It is also well established that MDM2 gene amplification can occur in diverse human malignancies that include soft tissue sarcomas, neuroblastoma, cancers of the brain, breast, ovary, cervix, lung, colon, prostate, and bone. Mechanisms underlying induction and stabilization of HIF-1α in normoxic conditions are less clearly understood. Our present data indicated that the normoxic expression level of HIF-1α was significantly higher (p&lt;0.0001) in LNCaP-MST as compared with LNCaP cells. When we treated the cells with the MDM2 inhibitor nutlin-3, the levels of HIF-1α was significantly down regulated. These results were further correlated with the differences in subcellular compartmentalization and degradation of HIF-1α. Findings from our lab shows the degradation rate of HIF-1α protein in both the nuclear and cytoplasmic compartments of LNCaP (non-transfected) and LNCaP-MST (MDM2 transfected) cells. In LNCaP cells, the degradation of HIF-1α protein occurs within 8 min in cytoplasmic (99%) and 10 min in nuclear compartments (75%). Whereas, in LNCaP-MST cells, the degradation rate of HIF-1α protein was slower even at 10 min of reoxygenation in both cytoplasmic (70%) and nuclear compartment (40%). Our results suggest that, MDM2 could play the role of master regulator in stabilizing HIF-1α even after reoxygenation. Further studies in this direction will shed more light on the in-depth mechanisms involved in the regulation of HIF-1α in MDM2 positive cancers. (This project was supported by The Royal Dames of Cancer Research Inc., Ft. Lauderdale, Florida).
Citation Format: Thanigaivelan Kanagasabai, Rohin Chand, AmyAman Kaur, Sivanesan Dhandayuthapani, Olena Bracho, Appu Rathinavelu. MDM2 stabilizes and induces HIF-1α levels during reoxygenation of cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1860.
Abstract 1870: Inhibition of Galectin-1 sensitizes oncogenic H-Ras to Rapamycin treatment
Authors: Michael James V. , Wurtzel Jeremy G T , Goldfinger Lawrence E. 
Published: July 2016

Abstract
Specific plasma membrane (PM) localization is essential for H-Ras signaling, and relies on post-translational modifications on the C-terminal targeting domain. H-Ras shuttles from the lipid ordered (Lo) domain to the lipid ordered/lipid disordered (Lo/Ld) border upon activation, which is dependent on Galectin-1. We have previously found that H-Ras, which is sequestered in the Lo domain by swapping the C-terminal targeting domain with the Lo-sequestering targeting domain of R-Ras, is deficient in MAPK signal propagation, while having no effect on PI3K activation, nor on H-Ras-driven tumor progression. We have further found that inhibition of PI3K with LY294002 inhibited tumor progression by H-Ras with or without Lo sequestration. Here we show that Lo sequestration of H-Ras attenuated, but did not completely block, H-Ras-induced mTOR signaling (S6kinase phosphorylation) despite similar activation of PI3K as H-Ras. Interestingly, MEK inhibition with U0126 diminished S6kinase phosphorylation by H-Ras, as well as by Lo-sequestered H-Ras. Here we demonstrate that H-Ras-driven allograft tumor growth in mice was substantially blunted upon treatment with mTOR inhibitor Rapamycin, and this effect of Rapamycin was further enhanced in tumors driven by Lo-sequestered H-Ras. Moreover, Rapamycin treatment ablated ERK phosphorylation in H-Ras tumors as well as in tumors with Lo-sequestered H-Ras (in which ERK phosphorylation was already greatly reduced). Together these findings indicate that Lo sequestration of H-Ras inhibits MAPK pathway activation, and that the MAPK pathway engages in crosstalk with mTOR pathways by regulating S6kinase phosphorylation downstream of H-Ras, whereas mTOR activity is required for H-Ras-induced MAPK signaling. These data further indicate that mTOR activation downstream of H-Ras is sufficient to drive tumorigenesis, but this pathway also requires H-Ras-MAPK activation. To recapitulate H-Ras Lo sequestration by the C-terminal targeting domain swap genetic model, we used a Galectin-1 inhibitor, OTX008, to disrupt H-Ras from transitioning between the Lo and Ld domains. OTX008 treatment alone inhibited H-Ras-driven allograft tumor growth to a similar extent as Rapamycin. However, a combination of OTX008 and Rapamycin resulted in nearly complete ablation of H-Ras-dependent tumor growth. These findings indicate that blockade of H-Ras targeting to the lipid ordered/disordered plasma membrane microdomain border, coupled with blockade of mTOR signaling, could provide a novel therapeutic approach to treat H-Ras-associated cancers.
Citation Format: James V. Michael, Jeremy G T Wurtzel, Lawrence E. Goldfinger. Inhibition of Galectin-1 sensitizes oncogenic H-Ras to Rapamycin treatment. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1870.
Abstract 1903: A genetic variation in microRNA target site of ETS2 is associated with clinical outcomes of paclitaxel-cisplatin chemotherapy in non-small cell lung cancer
Authors: JI WOONG SON , Lee Shin Yup , Kim Chang Ho , Park Jae Yong 
Published: July 2016

Abstract
Background: Recently, crosslinking, ligation, and sequencing of hybrids (CLASH) provided direct observation of transcriptome-wide miRNA-target pairs. The present study was performed to investigate the association of single nucleotide polymorphisms (SNPs) located in the miRNA target sites, which were experimentally verified by CLASH, with the clinical outcome of first line paclitaxel-cisplatin chemotherapy in advanced non-small cell lung cancer (NSCLC).
Methods: Eighty SNPs in miRNA target sites of cancer related genes selected from 18,500 miRNA:target interactions in CLASH data were investigated in 379 advanced NSCLC patients using a sequenom mass spectrometry-based genotype assay. Quantitative reverse transcription-polymerase chain reaction and luciferase assay were conducted to examine functional relevance of potentially functional SNPs in miRNA target sites.
Results: Of the 80 SNPs analyzed, 16 SNPs were significantly associated with the clinical outcome after chemotherapy. Among these, ANAPC1 rs3814026C&gt;T, ETS2 rs461155A&gt;G, SORBS1 rs7081076C&gt;A and POLR2A rs2071504C&gt;T were found to be significantly associated with both chemotherapy response and survival. Notably, the relative expression level of ETS2 was significantly associated with rs461155A&gt;G genotypes in both tumor and paired normal lung tissues (Ptrend = 4 × 10-7,and3×10-4,respectively). Consistently, a decreased expression of the reporter gene for the G allele of rs461155 compared with the A allele was observed by luciferase assay.
Conclusion: These findings suggest that the four SNPs, especially ETS2 rs461155A&gt;G, could be used as biomarkers predicting the response and survival of NSCLC patients treated with first-line paclitaxel-cisplatin chemotherapy.
Citation Format: SON JI WOONG, Shin Yup Lee, Chang Ho Kim, Jae Yong Park. A genetic variation in microRNA target site of ETS2 is associated with clinical outcomes of paclitaxel-cisplatin chemotherapy in non-small cell lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1903.
Abstract 1909: miR-30c blocks tumor cell homotypic aggregation and promotes anoikis to prevent breast cancer metastasis
Authors: Liu Xia , Yu Beijie , Chen Wenjing , Liu Huiping 
Published: July 2016

Abstract
Circulating tumor cells (CTCs) are considered the seeds of distant metastasis, and may be a predictor of poor prognosis in patients with metastatic breast cancers. Compared to single CTCs, clusters of multiple CTCs possess higher metastatic capacity in mouse breast cancer models, indicating that aggregation increases the survival of CTCs in the circulation. However, the molecular mechanisms by which clustered CTCs mediate tumor cell survival are unclear. In this study, we found that miR-30c, which has been identified as an anti-metastatic microRNAs, inhibited primary breast cancer cell homotypic aggregation in vitro by real-time monitoring. Interestingly, only aggregated but not single tumor cells can survive during culture. The death of dissociated primary tumor cells in vitro is caused by anoikis, a form of programmed cell death which is induced by detachment of anchorage-dependent cells from the surrounding extracellular matrix. Therefore, the role of miR-30c in anoikis was determined by culturing breast cancer cells in poly-hema coated plates. We found that miR-30c promoted anoikis, evaluated by Annexin-V staining and caspase3/7 activity. Mechanistic studies indicated that inhibition of Vimentin (Vim), a positive regulator of epithelial-to-mesenchymal transition (EMT), was required for miR-30c induced anoikis. In vivo, lung metastasis was inhibited by miR-30c after tail vein injection of miR-30c over-expressed cells. In summary, our findings provide the new insights into the mechanisms of miR-30c-mediated metastasis inhibition and suggest that targeting CTC clusters could be a novel strategy to prevent metastasis.
Citation Format: Xia Liu, Beijie Yu, Wenjing Chen, Huiping Liu. miR-30c blocks tumor cell homotypic aggregation and promotes anoikis to prevent breast cancer metastasis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1909.
Abstract 192: Targeting the protein kinase Hunk in HER2+ resistant breast cancer
Authors: Zambrano Joelle , Phelps-Polirer Kendall , Abt Melissa , Yeh Elizabeth 
Published: July 2016

Abstract
Despite the initial success of common HER2 inhibitors used in the clinic, including trastuzumab and lapatinib, HER2 positive (HER2+) breast cancer cells often acquire resistance through mechanisms that continue to remain poorly understood. A number of signaling molecules, which act downstream of HER2, including PI3K, Akt, JNK, and Hormonally-Upregulated Neu-associated Kinase (Hunk), are thought to contribute to resistance. Our recent studies indicate that targeting Hunk via shRNA in HER2+breast cancer cells that are sensitive to HER2 inhibitors (BT474) and cells that are resistant (JIMT) leads to increased caspase-3 activity in vitro and impaired tumor growth in vivo. In addition, we find that targeting Hunk not only sensitizes HER2+ cells to apoptosis, but also impairs autophagy, as indicated by decreased LC3BII expression, the lipidated form of LC3B which signifies active autophagy. Because elevated Hunk protein expression correlates with poorer prognosis, and since Hunk knockdown impairs autophagy, it is possible that Hunk acts to upregulate autophagy thereby contributing to the development of resistance. We have also found that the kinase JNK plays a role in resistance in HER2+ breast cancer cells, and demonstrate that JNK inhibition via a pan-JNK inhibitor impairs tumor growth of JIMT cells. We further show that co-targeting of JNK and HUNK results in greater cell death and significantly reduced tumor growth than either individual treatment in the JIMT-1, HER2+ resistant cell line, as well as a HER2+ line that was engineered to be lapatinib resistant, BT474-LR. Overall, our results indicate that targeting Hunk in HER2+ breast cancer not only alters autophagy and impairs tumor growth, but that co-targeting Hunk and JNK shows greater therapeutic effects on reducing tumor growth and inducing cell death.
Citation Format: Joelle Zambrano, Kendall Phelps-Polirer, Melissa Abt, Elizabeth Yeh. Targeting the protein kinase Hunk in HER2+ resistant breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 192.
Abstract 1977: Interaction between long noncoding RNA EZR-AS1 and SMYD3 promotes EZR expression in ESCC cells
Authors: Zhang Xiaodan , Huang Guo-Wei , Liao Lian-Di , Long Lin , Liao En-Min , Xu Li-Yan 
Published: July 2016

Abstract
EZR, a member of the ezrin-radixin-moesin (ERM) family, is involved in multiple aspects of cell migration by acting both as crosslinkers between the membrane, receptors and the actin cytoskeleton, and as regulators of signalling molecules that are implicated in cell adhesion, cell polarity and migration. SMYD3 (SET and MYND Domain-containing protein 3), an H3K4 histone methyltransferase, plays important roles in EZR gene transcription activity. However, the molecular mechanisms of how to recruit this epigenetic modifier to the EZR locus still remain ill-defined. In this study, we identify a natural antisense transcript (NAT) EZR antisense AS1 (EZR-AS1), which is transcribed from the opposite strand on the EZR gene locus, is involved in the SMYD3-mediated transcription of EZR. We find that EZR-AS1 is expressed in esophageal squamous cell carcinoma (ESCC) cells and is positive correlated with EZR expression in both ESCC cells and human ESCC tissues. Silenced EZR-AS1expression decreased EZR expression at both RNA and protein levels, resulting in an apparent inhibitory effect on cell motility. Overexpression of EZR in the EZR-AS1 depleted cells partly restored the mobility of cells. Through RNA-binding protein immunoprecipitation (RIP) assay and RNA pull down assay, we further demonstrate EZR-AS1 directly interacts with SMYD3, and it depends on an RNA/DNA hybrid formation. EZR-AS1 knockdown led to a significant decrease in both SMYD3 occupancy and histone H3K4me3 at the EZR promoter region, whereas EZR-AS1 overexpression results the opposite effects. Together, our results suggest a novel mechanism of interaction between a NAT EZR-AS1 and SMYD3 drives the EZR transcription in ESCC cells.
Citation Format: Xiaodan Zhang, Guo-Wei Huang, Lian-Di Liao, Lin Long, En-Min Liao, Li-Yan Xu. Interaction between long noncoding RNA EZR-AS1 and SMYD3 promotes EZR expression in ESCC cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1977.
Abstract 1980: Long-range regulation of HOTAIR identifies novel biomarkers of breast cancer outcome and suggests a role in genome instability
Authors: Milevskiy Michael J. , Al-Ejeh Fares , Saunus Jodi M. , Northwood Korinne S. , McCart-Reed Amy E. , Dray Eloise , Nephew Kenneth , Bailey Peter J. , Betts Joshua A. , Stone Andrew , Gee Julia M W , Shewan Annette M. , French Juliet D. , Edwards Stacey L. , Clark Susan J. , Lakhani Sunil R. , Brown Melissa A. 
Published: July 2016

Abstract
Long-range regulators of gene expression are emerging as promising new biomarkers and therapeutic targets for human diseases including cancer. As current breast cancer biomarkers have limited power for predicting disease progression and response to therapy, we have explored the potential of long-range regulators of non-coding RNAs to be useful in the prognostication of breast cancer. HOTAIR is a long non-coding RNA that is overexpressed, promotes metastasis, and is predictive of poor prognosis in breast cancer. Here we describe a long-range transcriptional enhancer of the HOTAIR gene that binds several hormone receptors and associated transcription factors, interacts with the HOTAIR promoter and augments HOTAIR transcription. This enhancer is dependent on FOXA1 and FOXM1 transcription factors and functionally interacts with a novel alternate HOTAIR promoter. Analysis of breast cancer gene expression data indicates that HOTAIR is co-expressed with FOXA1 and FOXM1 in HER2-enriched tumours, and these factors enhance the prognostic power of HOTAIR in this subtype of breast cancer. The combination of HOTAIR and FOXM1 also enables better predictions of response to endocrine therapy for ER+ breast cancer. FOXM1 is a member of the recently described chromosome instability module and consistent with this, the expression of HOTAIR and FOXM1 is associated with increased frequency of copy number alterations and somatic non-synonymous mutations. Our study corroborates the importance of enhancers in breast cancer, elucidates the transcriptional regulation of HOTAIR, suggests HOTAIR as a novel biomarker of patient response to endocrine therapy, and implicates HOTAIR in chromosome instability.
Citation Format: Michael J. Milevskiy, Fares Al-Ejeh, Jodi M. Saunus, Korinne S. Northwood, Amy E. McCart-Reed, Eloise Dray, Kenneth Nephew, Peter J. Bailey, Joshua A. Betts, Andrew Stone, Julia M W Gee, Annette M. Shewan, Juliet D. French, Stacey L. Edwards, Susan J. Clark, Sunil R. Lakhani, Melissa A. Brown. Long-range regulation of HOTAIR identifies novel biomarkers of breast cancer outcome and suggests a role in genome instability. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1980.
Abstract 1984: A regulatory triad consisting of BMI1, let-7i miRNA and ERK3 kinase in controlling cancer cell invasiveness
Authors: Elkhadragy Lobna , Chen Minyi , Long Weiwen 
Published: July 2016

Abstract
Extracellular signal-regulated kinase 3 (ERK3) is an atypical mitogen-activated protein kinase, whose biological activity is tightly regulated by its cellular abundance. ERK3 expression is upregulated in multiple cancer types, including head and neck cancers. Importantly, our recent finding revealed a critical role for ERK3 in promoting cancer cell invasiveness. However, little is known about the molecular regulation of ERK3 level in cancers. Here we identify a pathway which regulates ERK3 expression and cell invasiveness of head and neck cancers, comprising the oncogenic polycomb group protein BMI1 and the tumor suppressive miRNA let-7i.
To study the regulation of ERK3, we performed several loss-of-function and gain-of-function assays in head and neck cancer cell lines, including OECM1 and Fadu. Stable knockdown of BMI1 greatly reduced ERK3 protein level, whereas overexpression of BMI1 significantly increased ERK3 level, demonstrating a positive regulation of ERK3 by BMI1. Interestingly, we found that this regulation is mediated by the miRNA let-7i, whose transcription is repressed by BMI1, a component of the polycomb repressive complex 1. ERK3 mRNA is directly targeted by let-7i miRNA, which was demonstrated by the assay using a luciferase reporter containing the 3’UTR of ERK3 mRNA downstream of luciferase gene. Mutation of the let-7i binding site in ERK3 3’UTR abolished the negative regulation of let-7i on ERK3. Further, let-7i mimic decreased ERK3 expression and let-7i inhibitor increased ERK3 expression in head and neck cancer cells. To determine the functional significance of this regulation, we investigated their role in cancer cell invasiveness by performing trans-well migration/invasion assays. Importantly, ERK3 depletion abolished BMI1-induced increase in cancer cell migration and invasion, so was by let-7i mimic. Finally, we examined by immunohistochemistry the expression of both BMI1 and ERK3 in head and neck tumor specimens and found that ERK3 expression is positively correlated with BMI1.
All together, we unravel a novel regulatory mechanism for ERK3 by the BMI1/let-7i axis, which together controls head and neck cancer cell invasiveness.
Citation Format: Lobna Elkhadragy, Minyi Chen, Weiwen Long. A regulatory triad consisting of BMI1, let-7i miRNA and ERK3 kinase in controlling cancer cell invasiveness. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1984.