bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1826: IMI's CANCER-ID: Status of liquid biopsy standardization
Authors: Pantel Klaus , Terstappen Leon WMM , Baggiani Barbara , Krahn Thomas , Schlange Thomas
Published: July 2016

The CANCER-ID (www.cancer-id.eu) consortium was established in early 2015 with more than 30 partners as part of the Innovative Medicines Initiative (IMI), Europe's largest public-private partnership funded in equal parts by the European Union and the European Federation of Pharmaceutical Industries and Associations (EFPIA). The goal is to develop standard operating procedures (SOPs) and qualify respective mature technologies for pre-analytical sample handling, enrichment, isolation and analysis of Circulating Tumor Cells (CTCs), circulating free tumor DNA (ctDNA) and microRNAs (miRNAs) as novel blood-based biomarkers in cancer liquid biopsy, with a particular focus on Non-Small Cell Lung Cancer (NSCLC) and HER2-treatment refractory breast cancer. Over the 5-year period of the project, CANCER-ID will evaluate different technologies in the field, testing their clinical utility in multicenter clinical studies in support of regulatory approval of these devices. During the first year of the project, a number of different technology platforms have been implemented at several partner sites to establish SOPs with standardized blood samples spiked with selected cancer cell lines as standards. An important outcome of these studies are pre-analytical sample handling procedures, e.g. using different fixatives and tube systems as well as biobanking protocols that allow shipment and standardized long-term storage of liquid biopsy material. Most importantly, the knowledge generated within IMI's CANCER-ID will be used to establish and validate SOPs for the analysis of patient blood samples using different technologies, which represents a promising diagnostic tool holding the potential for optimized treatment decisions and therapeutic monitoring of cancer patients. The international exchange of human patient samples for research purposes poses a number of regulatory challenges linked to the different legal and ethical environments. The diverse national regulations for clinical samples and data require coordinated monitoring of interactions and guidance of the different partner organizations. As close collaboration with regulatory authorities is crucial for CANCER-ID to achieve its objectives, the consortium has established a contact with CDER/FDA via a Critical Path Innovation Meeting (CPIM). We will discuss the CANCER-ID approach to meet these many requirements and will present a matrix for coordinating such international efforts with stakeholders from different organizations.

Citation Format: Klaus Pantel, Leon WMM Terstappen, Barbara Baggiani, Thomas Krahn, Thomas Schlange. IMI's CANCER-ID: Status of liquid biopsy standardization. [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 1826.
Abstract 1838: Differential CXCL13 and CXCR5 expression mediated by down-regulation of select miRNAs in prostate cancer
Authors: Hinton Denise E. , Lillard James
Published: July 2016

Chemokines and their receptors are involved in the inflammatory response by attracting immune cells to sites of infection and wound healing. Chemokines have also been implicated in the progression of prostate cancer (PCa). We have previously shown a positive correlation between the expression of CXCL13 and CXCR5 proteins with PCa progression; in particular, the CXCR5 protein is expressed to a greater extent by prostate tumors isolated from African Americans than compared to those from European Americans. In the present study, we sought: (i) to determine whether CXCL13 and CXCR5 mRNA expression patterns correlated with overall and/or disease-free survival in PCa cases and (ii) to identify the possible molecular mechanisms that govern this association. CXCL13 and CXCR5 mRNA levels and PCa survival rates were evaluated using The Cancer Genome Atlas (TCGA) data sets and the CbioPortal, respectively. CXCR5 and CXCL13 mRNA expression did not correlate with prostate tumor stage or grade and was not associated with overall PCa survival. However, CXCR5 mRNA expression was significantly associated with poor disease-free survival. Gene map analysis using the UCSC Genome Browser revealed consensus sequence binding sites for four miRNA's (miR-192/215, miR-486, and miR-494) that could bind the 3’ UTR of the cxcr5 transcript. Our results showed that miR-494 was frequently deleted or down-regulated, confirming other reports that miR-494 is down-regulated in high-grade PCa cases. Of note, miR-494 also targets cxcr4 mRNA and suppresses migration of PCa cells. Others have shown that miR-486 is associated with p53 and PI3K-Akt pathways and miR-186 has a putative binding site in the 3’ UTR region of the cxcl13 transcript. This miRNA was down-regulated in PCa patients with initial metastasis. These results confirm our previous studies demonstrating a correlation between tumor expression and PCa progression and reveal new mechanisms of post-translational regulation of this chemokine receptor.

Citation Format: Denise E. Hinton, James Lillard. Differential CXCL13 and CXCR5 expression mediated by down-regulation of select miRNAs in prostate 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 1838.
Abstract 1846: Gene expression analysis of P-glycoprotein and LMWPTP in chronic myeloid leukemia cells treated with metformin and imatinib mesylate
Authors: Oliveira Ligia P. , Ferro Rafael G. , Galvão. Ana CSS
Published: July 2016

Chemotherapy is an effective treatment for many tumors; however, some tumors begin to exhibit a wide range of resistance after a few cycles of chemotherapy, which is known as phenotype of resistance to multiple drugs (multidrug resistance - MDR). The MDR phenotype arises through various mechanisms, but often is associated with an increased efflux of hydrophobic cytotoxic drugs. Therefore, the search for more selective and efficient antitumor compounds in the elimination of cancer cells boosts the research in oncology area. Studies of genes and pathways that are deregulated in tumors and that are essential to their growth may provide new strategies that lead to the development of therapeutic agents that target these pathways. Previous data from our research group have shown potent toxic activity of metformin, an oral hypoglycemic used for more than 30 years for the treatment of type II diabetes, on chronic myeloid leukemia cells (CML), potentiating the effects of imatinib mesylate, the standard chemotherapy used in the treatment of CML. CML cells treated with these agents showed changes in expression of proteins associated with the MDR phenotype. In this way, this project aimed to evaluate the expression of ABCB1 (Pgp) e ACP1 (LMWPTP) by Polymerase Chain Reaction in real time (qPCR) in LCM K562 cells and Lucena before and after treatment with metformin and/or imatinib mesylate, seeking to verify the effects of metformin, alone or in combination with Imatinib mesylate in the expression of these genes in Lucena in K562 cells. We found statistically significant differences in expression between treatments compared to control and between K562 and Lucena cells. It is possible to conclude that these genes may be associated with MDR phenotype.

Citation Format: Ligia P. Oliveira, Rafael G. Ferro, Ana CSS Galvão. Gene expression analysis of P-glycoprotein and LMWPTP in chronic myeloid leukemia cells treated with metformin and imatinib mesylate. [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 1846.
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

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

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<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

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

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>T, ETS2 rs461155A>G, SORBS1 rs7081076C>A and POLR2A rs2071504C>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>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>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

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 1914: CtBP1 and metabolic syndrome modulate cell adhesion and target multiple miRNAs in breast cancer cells
Authors: De Luca Paola , Farré Paula Lucía , Dalton Nicolás , Moiola Cristian Pablo , Porretti Juliana , Massillo Cintia , Scalise Georgina , De Siervi Adriana
Published: July 2016

Breast cancer is the leading cause of cancer death among women, after skin cancers. Although genetic susceptibility clearly influences cancer risk, non-inherited factors determine most of the differences in cancer risk across populations and among individuals. Metabolic syndrome (MeS) increases the incidence and aggressiveness of breast cancer. C-terminal binding protein 1 (CtBP1) is a co-repressor of tumor suppressor genes that is activated by low NAD+/NADH ratio. Previously, we found that both, CtBP1 and MeS modulated breast carcinogenesis and tumor growth using a MeS experimental mice model by chronically feeding animals with high fat diet (HFD). The most severe form of breast cancer is metastatic and at this stage the disease is often fatal. The aim of this work was to investigate CtBP1 and MeS role on cell adhesion, a key process for the establishment of metastasis. We found that CtBP1 protein diminished adhesion of MDA-MB-231 breast cancer cells. More important, serum from MeS animals diminished breast cancer cell adhesion compared to control serum. In addition nude mice fed with CD or HFD were subcutaneously injected with MDA-MB-231 breast tumor cells with CtBP1 depleted expression or control cells. We found that CtBP1 and MeS modulated expression of cell adhesion targets in the xenografts, such as Vimentin, Slug, ITGB4, Col17A1, FABP4 and PRSS2.

Interestingly, miRNA expression profile obtained by RNA from CtBP1 depleted or control xenograft tumors hybridization to GeneChip miRNA 4.0 (Affymetrix) identified 42 CtBP1 regulated miRNAs. We found 77 predicted miRNAs target genes up- and 30 genes down-regulated by this set of 42 differentially expressed miRNAs, using miRecords data base. Gene ontology (GO) analysis of all these genes revealed an enrichment of localization, metabolic processes, cellular process and biological regulation categories, among other biological functions. Examining processes within these GO functions; we found important categories overrepresented, such as cell cycle, cell communication, vesicle-mediated transport and primary metabolic process.

These results clearly show that CtBP1 and MeS diminish adhesion of breast cancer cells and suggest a key role for these components in the begining of metastasis. In addition, understanding CtBP1 and MeS regulation of miRNAs could be crucial as markers for the prevention, follow up and treatment of breast cancer.

Citation Format: Paola De Luca, Paula Lucía Farré, Nicolás Dalton, Cristian Pablo Moiola, Juliana Porretti, Cintia Massillo, Georgina Scalise, Adriana De Siervi. CtBP1 and metabolic syndrome modulate cell adhesion and target multiple miRNAs in breast 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 1914.
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

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

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

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

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.
Abstract 1993: IL-18 induces an anti-apoptotic effect through stabilizing COX-2 mRNA expression in leukemia
Authors: Chu Yu Yi , KO Chiung Yuan , Wang Ju Ming
Published: July 2016

Leukemia is a cancer of bone marrow or blood cells. The acute myeloid leukemia (AML) is a common subtype occurred in adults to result in death. Forcing malignant cells to undergo differentiation instead of killing cancer cells is one of AML therapeutic strategy. However, the AML patients with poor outcome due to drug resistance is still a challenge. In this study, the abundances of the pro-inflammatory cytokine IL-18 and its receptor (IL-18R) were found to be correlated with the occurrence of drug resistance in AML patients. Importantly, we found that IL-18 induced an anti-apoptotic effect on U937 and THP-1 cells. Cyclooxygenase 2 (COX-2) has been suggested to have an anti-apoptotic role in chemo-resistant cancer cells. IL-18 increased COX-2 expression through post-transcriptional regulation in differentiated U937 cells. We found that two RNA binding proteins, HuR and IMP-3, stabilized COX-2 mRNA expression. In addition, IL-18 induced their interaction and facilitated this complex shuttle from nucleus to cytoplasm. Subsequently, HuR and IMP-3 bound to COX-2 mRNA 3’UTR. Two chemical inhibitors of JNK and ERK1/2 attenuated IL-18-enhanced COX-2 transcripts and the shuttle of HuR from nucleus to cytoplasm. These results suggested that IL-18 stabilized COX-2 mRNA through the JNK- and/or ERK1/2-regulated HuR nucleocytoplasmic shuttling and contributed to an anti-apoptotic effect in AML.

Citation Format: Yu Yi Chu, Chiung Yuan KO, Ju Ming Wang. IL-18 induces an anti-apoptotic effect through stabilizing COX-2 mRNA expression in leukemia. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1993.
Abstract 2023: SOX4 is essential for PTEN-mediated prostate tumorigenesis in vivo
Authors: Bilir Birdal , Osunkoya Adeboye O. , Wiles W. Guy , Sannigrahi Soma , Lefebvre Veronique , Metzger Daniel , Spyropoulos Demetri D. , Martin W. David , Moreno Carlos S.
Published: July 2016

Prostate cancer is the most common cancer and the second leading cause of cancer mortality in American men, underscoring the significance of unraveling the molecular mechanisms involved in the initiation and progression of the disease. The sex-determining region Y-box 4 (SOX4) gene is overexpressed in many types of human cancers, including prostate cancer, suggesting that SOX4 plays a fundamental role in tumorigenesis. In this study, we demonstrate that SOX4 is critical for PTEN-mediated prostate cancer progression in vivo. We show that homozygous deletion of Sox4 in the adult prostate epithelium strongly inhibits tumor progression initiated by homozygous loss of the Pten tumor suppressor, demonstrating the key role of SOX4 in the development of prostate cancer. Homozygous deletion of Sox4 also reduces the activation of AKT and β-catenin in Pten-null mice, resulting in inhibition of an invasive cancer phenotype. We also show that SOX4 expression is induced by loss of PTEN, and that PI3K-AKT-mTOR signaling activity is critical for SOX4 expression, suggesting a positive feedback loop between SOX4 protein and PI3K-AKT-mTOR activity. Our findings indicate that SOX4 is a critical component of the PTEN-PI3K-AKT pathway in prostate cancer, suggesting that SOX4 may be a promising molecular target for novel combinatorial therapies for both primary and advanced prostate cancers.

Citation Format: Birdal Bilir, Adeboye O. Osunkoya, W. Guy Wiles, Soma Sannigrahi, Veronique Lefebvre, Daniel Metzger, Demetri D. Spyropoulos, W. David Martin, Carlos S. Moreno. SOX4 is essential for PTEN-mediated prostate tumorigenesis in vivo. [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 2023.
Abstract 2048: Pharmacodynamic biomarker studies of TRC105 anti-endoglin (CD105) antibody revealed anti-angiogenic activity associated with CD105 depletion
Authors: Cao Liang , Karzai Fatima , Apolo Andrea , Madan Ravi , Yu Yunkai , Gulley James , Figg William , Dahut William
Published: July 2016

Introduction: CD105 is involved in normal vascular development. It is over expressed on the surface of proliferating vascular endothelial cells and is implicated in tumor angiogenesis. In hypoxic conditions, CD105 is upregulated through induction of hypoxia-inducible factor 1-α. TRC105 is a chimeric IgG1 antibody specific for CD105 and the agent for this phase I trial.

Methods: 20 patients with metastatic prostate cancer were treated with TRC105 at six dose levels in a phase I trial. Blood samples were analyzed for CD105 antigen depletion, VEGF as a marker for systemic hypoxia, and PSA.

Results: Maximum tolerated dose of 20 mg/kg every two weeks was reached. Significant plasma CD105 reduction was observed at high dose levels. The reduction of CD105 was associated with induction of plasma VEGF. Ten patients had stable disease, and the reduction of CD105 is associated with PSA stabilization.

Conclusion: A significant induction of VEGF was associated with CD105 reduction at three high dose levels, suggesting the anti-angiogenic activity of TRC105. Exploratory analysis showed a tentative correlation between the reduced CD105 and a decreased PSA velocity, suggestive of potential antitumor activity of TRC105 in metastatic prostate cancer.

Citation Format: Liang Cao, Fatima Karzai, Andrea Apolo, Ravi Madan, Yunkai Yu, James Gulley, William Figg, William Dahut. Pharmacodynamic biomarker studies of TRC105 anti-endoglin (CD105) antibody revealed anti-angiogenic activity associated with CD105 depletion. [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 2048.
Abstract 2147: Overcoming mTOR resistance mutations with a new generation mTOR inhibitor
Authors: Rodrik-Outmezguine Vanessa S. , Okaniwa Masanori , Yao Zhan , Novotny Chris , McWhirter Claire , Banaji Arpitha , Won Helen , Wong Wai , Berger Mike , de Stanchina Elisa , Barratt Derek G. , Cosulich Sabina , Klinowska Teresa , Rosen Neal , Shokat Kevan M.
Published: July 2016

First generation mTOR inhibitors (rapalogs) have modest activity in some tumors. We sought to delineate the likely resistance mechanisms to existing mTOR inhibitors as a guide for next generation therapies. We identified FRB domain and kinase domain mutations as causing acquired-resistance to rapamycin and mTOR kinase inhibitor respectively.

Resistance to rapamycin was due to loss of binding of the FKBP12-rapamycin complex to the FRB domain of mTOR mutant protein. Resistance to the mTOR kinase inhibitor was not due to a gatekeeper mutation but rather to an increase in the intrinsic kinase activity of the mTOR kinase domain mutant. These and similar mutations have also been identified in tumors from untreated patients. These findings suggest that these mutations are gain of function mutants but also that tumors with these activating mutations will be intrinsically resistant to second generation mTOR kinase inhibitors.

We have designed a new class of mTOR inhibitor, RapaLink-1, that takes advantage of the juxtaposition of two drug binding pockets to create a bivalent interaction. This compound potently inhibits mTOR activation in cells with mTOR wild-type and overcomes resistance to existing first and second generation inhibitors. Moreover, intermittent dosing of RapaLink-1 effectively suppresses mTOR-dependent tumor growth in vivo. Thus, such third generation mTOR inhibitors could be useful for treating tumors with activating mutations of mTOR and for tumors with acquired-resistance to current mTOR inhibitors.[N.R. and K.S. contributed equally to this work.]

Citation Format: Vanessa S. Rodrik-Outmezguine, Masanori Okaniwa, Zhan Yao, Chris Novotny, Claire McWhirter, Arpitha Banaji, Helen Won, Wai Wong, Mike Berger, Elisa de Stanchina, Derek G. Barratt, Sabina Cosulich, Teresa Klinowska, Neal Rosen, Kevan M. Shokat. Overcoming mTOR resistance mutations with a new generation mTOR inhibitor. [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 2147.
Abstract 2177: Rutin enhances the antiproliferative effect of 5-FU and oxaliplatin in colon cancer cells
Authors: Nasiri Farnoud , Kismali Gorkem , Alpay Merve , Kosova Funda , Cakir Dilek Ulker , Sel Tevhide
Published: July 2016

Background: Rutin is a strong antioxidant molecule and it has advantageous over other flavonoids due to it is a nontoxic and nonoxidizable molecule. The concept of dual therapy of anti-cancer drugs with natural compounds has become a very promising approach in new strategy to treatment. The aim of this study was to evaluate the antiproliferative and apoptotic effects of rutin flavonoids and its efficacy in enhancing the anticancer effects of 5-FU (5-fluorouracil) and oxaliplatin against colon cancer cells.

Material and Methods: Caco-2 human colon cancer cells were treated with rutin and/or anticancer drugs (5-FU and oxaliplatin), cell viability and apoptotic parameters were examined. Cell viability was determined by MTT assay. Apoptotic markers (cleaved caspase 3, cleaved PARP and phosphor-Bad) were determined using specific enzyme-linked immunosorbent assay kits. Caspase-8 and caspase-9 were analyzed by colorimetric activity assay kit. DNA fragmentation was analyzed by agarose gel electrophoresis.

Results: The exposure Caco-2 human colon cancer cells to rutin, 5-FU and oxaliplatin resulted growth inhibition in a dose- and time-dependent manner. Cell viability assay shows that rutin inhibiting growth of Caco-2 cells at high concentrations (over 1000 μM). Combination with rutin markedly enhanced 5-FU and oxaliplatin growth inhibiting effects on Caco-2 cells. Results suggested that rutin had significant anticancer abilities; such rutin were capable of causing multifold decreases in the half maximal inhibitory concentration IC50 value of 5-FU and oxaliplatin. This flavonoid increased the levels of proteins associated with apoptotic cell death (phospho-Bad, cleaved caspase 3 and cleaved PARP) alone and combination. The activities of caspase 8 and caspase 9 were stimulated by the combination treatment of rutin and oxaliplatin then alone. DNA fragmentation was observed only on combination treatment.

Conclusions: Combined treatment with rutin and anticancer drugs (5-FU and/or oxaliplatin) is more effective than the individual treatments of drugs at inhibiting growth of Caco-2 cells. The use of lower 5-FU and oxaliplatin doses, with similar effects, could be also useful to reduce possible adverse effects of these drugs. However, further studies at molecular level are required to elucidate chemopreventive and chemotherapeutic effects of rutin on colon cancer.

Citation Format: Farnoud Nasiri, Gorkem Kismali, Merve Alpay, Funda Kosova, Dilek Ulker Cakir, Tevhide Sel. Rutin enhances the antiproliferative effect of 5-FU and oxaliplatin in colon 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 2177.