bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract A182: Targeting the topoisomerase I enzyme in cancer cells with acquired resistance to SN-38
Authors: Stenvang Jan , Jensen Niels Frank , Jandu Haatisha , Knudsen Steen , Agama Keli , Jensen Thomas , Hansen Anker , Jensen Peter Buhl , Pommier Yves , Cushman Mark , Brünner Nils
Published: May 2016

De novo or acquired resistance to anti-cancer drugs represents a major obstacle to successful treatment of cancer patients. In order to improve future cancer drug development we have established the DEN-50R cell line panel which when fully developed will consist of isogenic pairs of drug sensitive and resistant human cancer cell lines representing the 5 most common cancer types.

Based on the DEN-50R we have tested a number of novel anti-cancer drugs in this panel of cell lines. One class of drugs that includes the indenoisoquinolines LMP400 and LMP776, which have topoisomerase 1 inhibitory activity and recently passed phase 1 clinical studies, showed very interesting features in the cell line studies.

When tested in SN-38 (the active metabolite of irinotecan) -sensitive and -resistant isogenic colorectal cancer cell lines and breast cancer cell lines, LMP400 showed significant dose related cytotoxicity independent of the state of SN-38-resistance (Table). As these resistant cell lines have significant up-regulation of either the BCRP and/or mdr-1 protein, it can be concluded that the indenoisoquinoline LMP400 can target topoisomerase 1 enzyme without being a substrate for these drug efflux pumps.

We are now planning to initiate two clinical phase II studies (Simon's two stage design) with LMP400; one in irinotecan-failed metastatic colorectal cancer patients and one in late stage metastatic breast cancer patients. Patients for these studies will be preselected based on an LMP400-responsiveness profile we generated by gene expression data where associations between gene expression profiles and growth inhibition were compared in a panel of cell lines exposed to LMP400. A second step included filtering the identified gene expression profile against mRNA expression from a collection of 3200 human tumors. Only genes being differentially expressed in the clinical tumor material were retained in the model.

Sensitivity to LMP776 and LMP400 of human colorectal and breast cancer cell lines with acquired resistance to SN-38

Cell lineCross-resistantSensitiveHCT116-SN-38*LMP400; LMP776HT29-SN-38LMP776LMP400LoVo-SN-38LMP776LMP400MDA-MB-231- SN-38LMP776LMP400MCF-7- SN-38LMP776; LMP400* HCT-116-SN38 resistant cells have gained a mutation in the binding site of SN38 and LMP400 during acquisition of SN-38 resistance.

Citation Format: Jan Stenvang, Niels Frank Jensen, Haatisha Jandu, Steen Knudsen, Keli Agama, Thomas Jensen, Anker Hansen, Peter Buhl Jensen, Yves Pommier, Mark Cushman, Nils Brünner. Targeting the topoisomerase I enzyme in cancer cells with acquired resistance to SN-38. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A182.
Abstract CN07-01: Using CTCs to improve outcomes in SCLC: Single cell profiling, ex vivo cultures, and mouse models
Authors: Dive Caroline
Published: May 2016

There is increasing agreement that minimally invasive biomarkers to monitor patients receiving targeted therapies have significant potential to facilitate personalized medicine. The rarity and heterogeneity of CTCs make them a technically demanding source of biomarkers but one that holds great promise as the technical challenges are met. Marker independent technology platforms are in development which will better assess CTC subpopulations, but none so far have been fully validated for clinical trial use and treatment decision-making. CTC enumeration with CellSearch (EpCam and cytokeratin positive CTCs) has prognostic significance in many epithelial tumors, and in diseases with prevalent CTCs such as small cell lung cancer (SCLC) the dynamic range of CTC number allows pharmacodynamic evaluation. Biomarkers based on multiplex protein analysis and FISH are also evaluable in CTCs if sufficient can be detected. Examples of CTC based biomarkers in clinical trials will be presented.

Developments in single CTC isolation and genomic profiling are increasingly reported by allowing insight to the biology of invasive tumor cells, including cellular co-expression of cancer specific mutations. I will present our progress in DNA profiling of single SCLC CTCs, and how this approach may inform on inter- and intratumor heterogeneity, and via serial sampling, on mechanisms of drug resistance. Most recently, we developed lung cancer patient CTC derived mouse models (termed CDX). SCLC CDX models faithfully recapitulate patient drug responses and will be useful to test novel therapeutics. CDX are generated at patient presentation and for those patients who first respond and then relapse with drug resistant disease, the relapse blood sample can be used to generate a paired CDX. Alongside single CTC profiling, the CDX approach will allow a comprehensive analysis of acquired drug resistance to chemotherapy, the discovery of new drug targets and testing of targeted therapies and drug combinations. I will also describe recent research exploring vasculogenic mimicry (VM) in SCLC, whereby tumor cells adopt endothelial characteristics and form vessels to increase nutrient and oxygen delivery to tumor independent of host vasculature. SCLC VM may explain the prevalence of CTCs and the early dissemination of this dismal disease and VM pathways may provide novel therapeutic targets.

Finally, I will present a case report on a NSCLC patient whose blood sample contained no CellSearch CTCs yet generated a CDX model. CTC filtration of this patients blood revealed a high proportion of mesenchymal CTCs consistent with Epithelial to Mesenchymal Transition (EMT).

Citation Format: Caroline Dive. Using CTCs to improve outcomes in SCLC: Single cell profiling, ex vivo cultures, and mouse models. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr CN07-01.
Abstract CN07-04: Dissecting the multicellular ecosystem of metastatic melanoma by single-cell RNA-seq
Authors: Tirosh Itay , Izar Benjamin , Prakadan Sanjay M. , Wadsworth Marc H. , Treacy Daniel , Trombetta John J. , Lu Diana , Rotem Asaf , Lian Christine , Murphy George , Cohen Ofir , Allen Eli van , Bertagnolli Monica , Genshaft Alex , Hughes Travis K. , Ziegler Carly G. K. , Kazer Samuel W. , Gaillard Aleth , Kolb Kellie E. , Valbuena1 Judit , Yoon Charles , Rozenblatt-Rosen Orit , Shalek Alex K. , Regev Aviv , Garraway Levi
Published: May 2016

A single tumor is composed of malignant cells in diverse genetic and epigenetic states, and this diversity presents a significant barrier for targeted therapies. Furthermore, diverse non-malignant cells, such as immune, fibroblasts and endothelial cells shape the tumor microenvironment and are emerging as important drug targets. However, the diversity of cellular states among malignant and non-malignant cells within any tumor remains poorly understood. To begin to address these challenges we applied single-cell RNA-seq to profile >3,000 single cells isolated from 16 fresh human melanomas, and characterized distinct cell types and cell states. We found that malignant cells within the same tumor display transcriptional heterogeneity associated with multiple biological processes. In particular, subpopulations of cells in treatment-naïve tumors expressed a transcriptional program associated with resistance to RAF/MEK inhibition, and these were enriched in post-relapsed samples. Among several non-malignant cell types that were identified we focused on tumor-infiltrating T-cells and identified multiple profiles of exhaustion which differed among patients and could be linked to prior immunotherapies. Finally, we used our single cell–derived profiles of cell types within melanoma to deconvolve publicly available bulk tumor profiles and infer interactions between cells in the tumor microenvironment. This work demonstrates the capacity of single cell transcriptomics to offer new insights with implications for both targeted and immune therapies and will be broadly applicable to other tumor types.

Citation Format: Itay Tirosh, Benjamin Izar, Sanjay M. Prakadan, Marc H. Wadsworth II, Daniel Treacy, John J. Trombetta, Diana Lu, Asaf Rotem, Christine Lian, George Murphy, Ofir Cohen, Eli van Allen, Monica Bertagnolli, Alex Genshaft, Travis K. Hughes, Carly G. K. Ziegler, Samuel W. Kazer, Aleth Gaillard, Kellie E. Kolb, Judit Valbuena1, Charles Yoon, Orit Rozenblatt-Rosen, Alex K. Shalek, Aviv Regev and Levi Garraway. Dissecting the multicellular ecosystem of metastatic melanoma by single-cell RNA-seq. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr CN07-04.
Abstract PR01: Structural mechanism of sequence-specific 5-methylcytosine (5mC) recognition by AP-1 transcription factors
Authors: Hong Samuel , Cheng Xiaodong
Published: May 2016

DNA methylation is generally known to inhibit transcription factor binding. However, this notion has been challenged with identifications of several transcription factors that preferentially activate methylated promoters in specific sequences. AP-1 transcription factor (c-Jun/c-Fos heterodimer), a master regulator of a wide range of cancer-related genes, has been shown to bind a novel consensus sequence (5'-TGACTCG-3') in CpG methylation-dependent manner. In the fluorescence polarization-based DNA binding assay, both c-Jun/c-Fos heterodimer and c-Jun/c-Jun homodimer preferentially bind DNA containing the methylated consensus sequence compared to DNA with unmethylated sequence. Using a homodimer of c-Jun/c-Jun basic leucine zipper (bZIP) DNA binding domain, we have solved the X-ray structure of AP-1 in complex with an oligonucleotide containing the methylated consensus sequence. The structure reveals that a common motif conserved among bZIP proteins is involved in the specific recognition of methylated CpG by one monomer and TpG by the other monomer. Like 5mC, thymine (5-methyluracil) contains a methyl group at C5 position, and the similarity imposed by the methyl C5 preserves key structural features of DNA sequence recognized by AP-1. Our in vitro finding resonates with previous studies of AP-1-like transcription factors that preferentially bind methylated response elements. In light of such evidence, our characterization of AP-1 as a sequence-specific 5mCpG and TpG reader suggests a novel way in which DNA methylation may function to activate transcription in a methylation-dependent manner.

Citation Format: Samuel Hong, Xiaodong Cheng. Structural mechanism of sequence-specific 5-methylcytosine (5mC) recognition by AP-1 transcription factors. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr PR01.
Abstract A36: Nuclear NUAK1 function on cancer cell survival, independently of LKB1 and p53
Authors: Palma Mario , Peña Daniela , Venturelli Katherine , Castro Ariel F.
Published: May 2016

Cancer cells evade cell death during tumor progression under metabolic stress. However, molecular mechanisms involved in cellular adaptation to this stress are still poorly understood. NUAK1, a serine threonine kinase of the AMPK catalytic subunit family, has been involved in tumor cell survival, but its regulation and function under metabolic stress remain unclear. For this study, we have used mouse embryo fibroblasts (MEFs) and cancer cells to better understand the role of NUAK1 in cell survival. We found that NUAK1 mRNA and protein levels increase in response to metabolic stress, suggesting a transcriptional regulation of NUAK1 expression. In addition, we found that NUAK1 is expressed almost exclusively in the nucleus of MEFs and several cancer cell lines. Because of the nuclear localization of NUAK1 and previous studies showing that NUAK1 regulates p53 transcription factor, we investigated whether p53 is necessary for NUAK1´s effect on cell survival. By using p53ER/ER MEFs that can be reversibly and rapidly switched between p53 wild-type and knockout states, we demonstrated that NUAK1 can promote cell survival in a p53-independent cellular context. Furthermore, NUAK1 was involved in cell survival of p53-null H1299 lung cancer and p53-null HCT116 colorectal cancer cells under metabolic stress. In parallel to the increase of NUAK1 expression, we found that NUAK1 is phosphorylated. LKB1 kinase is known to phosphorylate NUAK1, increasing NUAK1 kinase activity. However, shRNA-mediated depletion of LKB1 or mutation of the NUAK1 residue phosphorylated by LKB1 did not affect NUAK1 function on cell survival. By bioinformatics analysis of NUAK1 protein sequence, we found several sites predicted to be phosphorylated by JNK and p38. Future studies will determine whether JNK or p38 is involved in NUAK1 phosphorylation, and also evaluate whether NUAK1 nuclear localization is directly related with tumor progression in a p53-independent manner. Overall, our results showed that NUAK1 is transcriptional and post-translational regulated in response to metabolic stress, and that the effect of NUAK1 on cell survival is independently of LKB1 and p53.

Funded by FONDECYT 1120923

Citation Format: Mario Palma, Daniela Peña, Katherine Venturelli, Ariel F. Castro. Nuclear NUAK1 function on cancer cell survival, independently of LKB1 and p53. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr A36.
Abstract A39: Sall2 tumor suppressor protein is a novel Casein Kinase 2 target
Authors: Hermosilla Viviana , Rabalski Adam , Gyenis Laszlo , Litchfield David W. , Pincheira Roxana
Published: May 2016

Sall2 is a transcription factor with critical roles during development, including neurogenesis and eye formation. Importantly, Sall2 has also been associated with cancer, although its contribution to the disease remains controversial. Sall2 is down-regulated in several tumor types and its activity promotes cell cycle arrest and cell death. Despite its role as a tumor suppressor, little is known about its regulation.

In order to identify regulatory mechanisms of Sall2 protein, bioinformatic analysis were performed. Preliminary data lead to the identification of several putative phosphorylation sites within a potential and highly conserved PEST motif. All of these sites match the consensus sequence for Casein Kinase 2 (CK2)-mediated phosphorylation. CK2 is a ubiquitous and constitutively active kinase whose activity is increased in cancer cells. CK2 promotes both cell survival and proliferation by regulating stability, activity and/or subcellular localization of its target proteins, including tumor suppressors.

With the aim of determining whether Sall2 is regulated by CK2, several approaches such as pharmacological inhibition of CK2, mass spectrometry analysis, immunofluorescence, immunoprecipitation, in vitro kinase assays and site directed mutagenesis were used.

Our results indicate that Sall2 is a CK2 target in vitro. In cells, CK2 interacts with Sall2 and triggers the ubiquitination and subsequent degradation of Sall2 in a proteasome-mediated fashion. Moreover, phosphorylation of Sall2 at S763 and T778 was greatly reduced under CK2 inhibition, suggesting that these are the most important CK2-dependent phospho-sites in vivo.

In conclusion, we have identified Sall2 as a new CK2 target. Our results support a novel regulatory mechanism for Sall2 degradation and provide new insights into the means by which CK2 promotes cell survival and proliferation, two characteristic requirements of cancer cells.

FUNDING: FONDECYT1151031, CIHR

Citation Format: Viviana Hermosilla, Adam Rabalski, Laszlo Gyenis, David W. Litchfield, Roxana Pincheira. Sall2 tumor suppressor protein is a novel Casein Kinase 2 target. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr A39.
Abstract A47: Expression of oncogenic alleles induces multiple blocks to HCMV infection
Authors: Xu Shihao , Schafer Xenia , Munger Joshua
Published: May 2016

Human cytomegalovirus (HCMV) poses serious threats to newborns and immune-compromised individuals. In contrast to most viruses studied, HCMV is unable to replicate in cancerous cells, but the mechanisms of this restriction are unclear. We explored these mechanisms by determining whether defined oncogenic alleles including the simian virus 40 (SV40) T antigens and oncogenic HRas G12V could inhibit HCMV replication. Our results show that the expression of SV40 T antigens blocks HCMV replication. In contrast, the replication of a related herpes virus, herpes simplex virus I, is not impacted by T antigen expression. The earliest restriction of HCMV infection involves a block of viral entry, as T antigen expression prevents viral tegument protein delivery. Subsequently we find that T antigen expression reduces the accumulation of platelet-derived growth factor receptor (PDGFR) alpha, a host protein that facilitates HCMV entry. Viral entry, gene expression and DNA replication could be partially rescued by PDGFR alpha over-expression. However, production of infectious viral progeny is not restored by PDGFR alpha over-expression in T antigen-expressing fibroblasts. Analysis of viral gene expression indicates that the accumulation of immediate early 2 (IE2), the major viral transcriptional activator, is inhibited by T antigen expression. Restoration of IE2 expression markedly increases downstream HCMV gene expression but not virion production, suggesting additional viral restrictions induced by T antigen. Similarly, PDGFR alpha over-expression in Hela cells markedly increases HCMV gene expression and DNA replication but not virion production. Combined, our results indicate that oncogenic alleles induce multiple restrictions to HCMV infection.

Citation Format: Shihao Xu, Xenia Schafer, Joshua Munger. Expression of oncogenic alleles induces multiple blocks to HCMV infection. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr A47.
Abstract B13: In vitro animal cancer model for assessment of sulfated polysaccharides extract of Ulva lactuca as colon cancer therapeutic and chemopreventive agents
Authors: Abu-Serie Marwa , Demellawy Maha El , El-Sayed Mohamed , El-Rashidy Fatma
Published: May 2016

Purpose: Sulfated polysaccharides extract of Ulva lactuca (ULSP) was in vitro assessed as probable chemopreventive and therapeutic agents for colon cancer (CC) comparing with currently used chemotherapy (5-fluorouracil “5-FU” and mitomycin-C “MMC”) using our newly developed in vitro transformed cancerous rat colon epithelial cells.

Methods: The present study used an in vitro animal CC model as it resembles human multistage of chromosomal instability type of CC that is located mainly at distal colon. Hence, this model was used for evaluating ULSP by the detection of cytotoxicity against normal and cancerous colon cells and antimetastatic potency. Besides, ULSP efficacy as CC chemopreventive agent was assessed through the detection of CC progression markers; loss of heterozygosity of deleted in colorectal cancer (DCC) and p53, aldehyde dehydrogenase (ALDH) 1B1 expression, activities of ornithine decarboxylase (ODC), cyclooxygenase-2 (COX-2) and alkaline phosphatase (ALP), cellular redox state (Eh), doubling time, and mucin level.

Results: The apoptosis-dependent anticancer activity of ULSP (4.9 µg/ml) was revealed at all stages of cancerous distal colon epithelial cells by the formation of apoptotic bodies and DNA fragmentation (above 43%) without affecting normal colon epithelial cells. Also, its inhibitory effect on the invasion of cancerous epithelial and Caco-2 cells (above 49%) was stronger than that of 5-FU+MMC (about 11%). Cells were treated with ULSP before exposure to CC inducers and they showed normal tumor suppressor genes (DCC and p53), normal expression of ALDH1B1, and normal activity of ODC (0.03±0.0004 U/μg) and null activity of COX-2. In addition, their normal pattern of proliferations (doubling time 3 days and Eh 252.7±2.12 mV) and differentiation was confirmed by the maintenance of ALP activity (0.46±0.01 mU/μg) and the secretion of mucin (29.2±0.2 μg/ml) compared to normal and cancerous colon epithelial cells.

Conclusion: Our innovative in vitro model of colon cancer have proved that ULSP could have induced apoptosis at all stages of cancerous colon cells, inhibited cancerous cells to metastasize and blocked CC progression without effect on normal epithelial cells comparable to chemotherapy.

Citation Format: Marwa Abu-Serie, Maha El Demellawy, Mohamed El-Sayed, Fatma El-Rashidy. In vitro animal cancer model for assessment of sulfated polysaccharides extract of Ulva lactuca as colon cancer therapeutic and chemopreventive agents. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr B13.
Abstract B27: Involvement of c-MET signaling in BCRP/ABCG2 activation and resistance to doxorubicin and photodynamic therapy
Authors: Choi Bo-hyun , Jung Kyeong-Ah , Kwak Mi-Kyoung
Published: May 2016

Overexpression of BCRP/ABCG2, a xenobiotic efflux transporter, is related to anticancer drug resistance in tumors. Proto-oncogene c-MET induces cancer cell proliferation, motility, and survival, and its aberrant activation was found to be a prognostic factor in advanced ovarian cancers. In this study, we demonstrate the potential cross-resistance of doxorubicin-resistant ovarian cancer cells to the pheophorbide a (Pba)-based photodynamic therapy (PDT) and suggest c-MET and BCRP/ABCG2 overexpression as an underlying molecular mechanism. The doxorubicin resistant A2780 cell line (A2780DR) showed enhanced resistance to PDT cytotoxicity with decreased level of reactive oxygen species generation and Pba accumulation than A2780. In microarray analysis, BCRP/ABCG2 is the sole drug transporter which was upregulated in A2780DR. The incubation with the BCRP/ABCG2 specific inhibitor reversed the both Pba-PDT and doxorubicin resistance in A2780DR. Importantly, we identified that the expression of c-Met, which is an oncogene activating PI3K signaling, was increased in A2780DR and the inhibition of PI3K/AKT or c-MET repressed resistance to doxorubicin and PDT. Finally, we showed that the pharmacological and genetic inhibition of c-MET diminished levels of BCRP/ABCG2 in A2780DR. Collectively, our results provide evidence that c-MET overexpression is linked to BCRP/ABCG2 activation and this mechanism leads to crossresistance to both chemotherapy and PDT.

Citation Format: Bo-hyun Choi, Kyeong-Ah Jung, Mi-Kyoung Kwak. Involvement of c-MET signaling in BCRP/ABCG2 activation and resistance to doxorubicin and photodynamic therapy. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr B27.
Abstract B29: Characterization of the regulatory feedback program components after HER2 inhibition in breast cancer cells
Authors: Sakur Nareeza , Fink Marc
Published: May 2016

HER2 overexpression drives tumorigenesis in a subset of breast cancers and pharmacological inhibition of this receptor tyrosine kinase is currently used in the clinic for these patients. While all of the cancer cells within the tumor are dependent on HER2 signaling, treatment with HER2 inhibitors results in the establishment of two separate populations; cells that undergo apoptosis and cells that survive. The mechanisms underlying the decision-making processes are not well understood. A proposed mechanism for establishing resistance within a population involves induction of signaling components that may allow for bypass of HER2. Induction of these genes is dependent on AKT inhibition and subsequent activation of the FOXO family of transcription factors. The best-studied adaptive signaling components relevant to HER2-positive breast cancer are HER3, IGF1R, and IR. The involvement of these signaling components in acute adaptive resistance to HER2 inhibition was studied in the HER2-positive BT474 cell line. Lapatinib increased HER3 protein levels in a concentration dependent manner, however, at higher concentrations this effect was reversed. In contrast, inhibition of PI3K increased HER3 levels even at higher concentrations. Time course analysis of HER3 induction demonstrated strong regulation of protein levels at 8 hours post addition of inhibitor. Ongoing work is focused on determining whether these findings are generally applicable to the adaptive gene response. The results reported here provide a framework to model the effects of HER2 inhibition on apoptosis and survival in breast cancer cells.

Citation Format: Nareeza Sakur, Marc Fink. Characterization of the regulatory feedback program components after HER2 inhibition in breast cancer cells. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr B29.
Abstract B36: Orally active low molecular weight heparin conjugates as a tumoral lymphangiogenesis inhibitor
Authors: Choi Jeong Uk , Byun Youngro
Published: May 2016

Heparin has been reported as not only an anticoagulant drug but also as a tumoral angiogenesis inhibitor by blocking tumor growth factors including VEGF, bFGF. Low molecular weight heparin conjugated with 4 pairs of deoxycholic acid (LHbisD4) was newly synthesized to enhance the oral absorption and efficacy. As the tumoral lymphangiogenesis is being considered to play the most important role in tumor metastasis, the purpose of this study is to evaluate whether LHbisD4 could suppress the tumoral lymphangiogenesis as well as the incidence of metastasis. To evaluate the inhibition effect of LHbisD4 on VEGF-C induced lymphangiogenesis, several in vitro experiments using human dermal lymphatic endothelial cell(HDLEC) was carried out. The inhibition effect of LHbisD4 on VEGF-C induced VEGFR-3 phosphorylation was evaluated by western blot. Proliferation assay, tubular formation assay, spheroid based sprouting assay were also done to evaluate the efficacy of LHbisD4 on suppressing lymphagiogenesis using HDLEC. For in vivo test, orthotopic breast cancer metastasis model was used and the incidence of metastasis to distant organs was evaluated. We found that VEGF-C induced phosphorylation of VEGFR3 which is the most important signaling cascade to initiate the lymphangiogenesis was highly inhibited in the presence of LHbisD4. Also through other in vitro experiment such as proliferation assay, tubular formation assay, spheroid based sprouting assay, LHbisD4 was proven to be effective enough to suppress VEGF-C induced lymphangiogenesis. In orthotopic model, 10mg/kg/day dose of LHbisD4 was administered orally and we found the incidence of metastasis is highly decreased compared with control group. Especially, weight and volume of the lymph node is highly decreased in case of drug treated group, which means the lymphatic metastasis is significantly suppressed. Through this study, we demonstrate that our novel heparin conjugate could be a good candidate for targeting VEGF-C induced lymphangiogenesis as well as an effective anti-metastatic agent.

Citation Format: Jeong Uk Choi, Youngro Byun. Orally active low molecular weight heparin conjugates as a tumoral lymphangiogenesis inhibitor. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr B36.
Abstract B38: A novel (+)-dehydroabietylamine derivative shows activity against triple-negative breast cancer cells
Authors: Gautam Lekh Nath Sharma , Ling Taotao , Rivas Fatima
Published: May 2016

In both well developed and developing countries, breast cancer remains the leading cause of cancer-related death among women. Very few effective treatments are available to its subtype triple negative breast cancer (TNBC), being unresponsive to estrogen therapy. A phenotypic screening was performed on terrestrial natural products against the human breast carcinoma cell lines MDA-MB-231, MA11, and MCF-7 targeting new chemical scaffolds to TNBC. Further studies were focused on abietanes natural products that preferentially inhibited proliferation of triple-negative MDA-MB-231 cells over estrogen receptor–positive cells. The activity of the abietane carnosol prompted to generate a focus library from the readily available (+)-dehydroabietylamine. The lead compound (+)-pentafluoro sulfonamide derivative of dehydroabietylamine displayed a promising EC50 of 9.0 μM against MDA-MB-231 and our mechanistic studies indicate it induced apoptosis, which was associated with activation of caspase-9 and -3 and the cleavage of PARP. Current progress towards this promising therapeutic area will be discussed.

Citation Format: Lekh Nath Sharma Gautam, Taotao Ling, Fatima Rivas. A novel (+)-dehydroabietylamine derivative shows activity against triple-negative breast cancer cells. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr B38.
Abstract IA11: Tumor dormancy and recurrence
Authors: Chodosh Lewis A.
Published: May 2016

For many types of human cancer, residual tumor cells remain following treatment that are not detected by conventional clinical testing. These cells have the ability to survive in a presumed dormant state within tissues for up to 20 years, either as solitary cells or as micrometastases. Ultimately, residual cells re-emerge from this latent state and resume growth, leading to cancer recurrence. Despite the unrivaled clinical importance of these aspects of breast cancer progression, the mechanisms underlying them are largely unknown. Since dormant residual tumor cells likely constitute the reservoir from which recurrent cancers arise, the lack of therapeutic approaches specifically targeted against these cells as well as our lack of understanding about their biology constitute major obstacles to the successful treatment of human cancers. As such, the development of targeted therapies designed to block pathways on which residual tumor cells depend for survival and growth would represent an attractive approach to preventing cancer recurrence. Consequently, elucidating the pathways that underlie dormant tumor cell survival and recurrence is a critical priority in cancer research.

Citation Format: Lewis A. Chodosh. Tumor dormancy and recurrence. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr IA11.
Abstract IA26: Long-term single cell quantification: New tools for old questions
Authors: Schroeder Timm
Published: May 2016

Stem cell systems are highly complex and dynamic, and consist of large numbers of different cells expressing many molecules. Despite intensive research, many long-standing questions in stem cell research remain disputed. One major reason is the fact that we usually only analyze populations of cells - rather than individual cells - at very few time points of an experiment. Tracking of individual cells would be an extremely powerful approach to improve our understanding of molecular cell fate control. We are therefore developing imaging systems to follow the fate of individual cells over many generations. We program new software to help recording and displaying the divisional history, position, properties, interaction, etc. of all individual cells over many generations. Our approaches also allow continuous long-term quantification of protein expression or activity in individual living cells. The resulting novel kind of continuous quantitative single cell data is used for the generation of improved models describing the molecular control of stem cell fates. I will discuss how we try to find answers for long standing questions in stem cell research.

Citation Format: Timm Schroeder. Long-term single cell quantification: New tools for old questions. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr IA26.
Abstract IA36: Signatures of mutational processes
Authors: Stratton Mike
Published: May 2016

All cancers are caused by somatic mutations. However, the processes underlying the genesis of somatic mutations in human cancer are remarkably poorly understood. Recent large-scale cancer genome sequencing initiatives have provided us with new insights into these mutational processes through the mutational signatures they leave on the cancer genome. In this talk I will review the mutational signatures found across cancer and consider the underlying mutational processes that have been operative.

Citation Format: Mike Stratton. Signatures of mutational processes. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr IA36.
Abstract A06: Characterization of a novel p53-regulated embryonic stem cell-specific lncRNA that safeguards pluripotency
Authors: Jain Abhinav K. , Akdemir Kadir C. , Xi Yuanxin , McCarthy Ryan L. , Allton Kendra , Aronow Bruce , Lin Chunru , Li Wei , Yang Liuqing , Barton Michelle C.
Published: May 2016

Recent work shows that long non-coding RNAs (lncRNAs), transcribed from 80% of our genomes, play important roles in gene regulation and are associated with disease pathogenesis. However, there is a broad gap in our knowledge regarding any contributions of lncRNAs in mediating p53 response or regulating stem cell state. Here, we report our discovery of lncRNAs that fine-tune p53's transcriptional outcomes in differentiating human embryonic stem cells (hESCs). By integrating RNA-Seq with p53 ChIP-Seq analyses along with active or repressive histone marks of hESCs undergoing differentiation, we defined a high-confidence set of 40 lncRNAs that are p53 transcriptional targets. We found that p53 directly regulates nuclear lncRNAs, which are either activated (HOTAIRM1) or repressed (lncPRESS-1) during hESC differentiation. Single-cell gene expression analysis during lineage-specific hESC differentiation revealed that lncPRESS-1 is a novel pluripotency-specific, p53-regulated transcript with expression tightly correlated with OCT4, NANOG and PRDM14. Loss-of-function of lncPRESS-1 results in diminished pluripotency and spontaneous differentiation of hESCs. In addition, mass cytometry-based multi-parametric analysis at a single cell level revealed that depletion of lncPRESS-1 results in overall loss of hESC pluripotency with marked induction of an ectoderm specific gene-signature. Further, we show that lncPRESS-1 physically interacts with SIRT6 and restrains its chromatin localization to maintain high levels of histone H3 Lys-56 (H3K56) and Lys-9 (H3K9) acetylation at promoters of pluripotency genes to sustain a chromatin architecture that favors pluripotency. In summary, we describe a novel pluripotency-specific lncRNA that safeguards the hESC state by disrupting SIRT6 activity. Since lncPRESS-1 is repressed by p53, we believe that cancers that harbor mutant TP53 will have robust expression of this lncRNA. Further investigations are underway to determine the disease relevance of lncPRESS-1 and correlation with p53-mutations.

Citation Format: Abhinav K. Jain, Kadir C. Akdemir, Yuanxin Xi, Ryan L. McCarthy, Kendra Allton, Bruce Aronow, Chunru Lin, Wei Li, Liuqing Yang, Michelle C. Barton. Characterization of a novel p53-regulated embryonic stem cell-specific lncRNA that safeguards pluripotency. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr A06.
Abstract A12: miR-346 and miR-138 competitively regulate hTERT in GRSF1- and AGO2-dependent manners, respectively
Authors: Tang Hua
Published: May 2016

miRNAs typically downregulate the expression of target genes by binding to their 3'UTR, and dysregulation of miRNAs may contribute to tumorigenesis. Here, we found that miR-346 and miR-138 competitively bind to a common region in the 3'UTR of hTERT mRNA and have opposite effects on the expression and function of hTERT in human cervical cancer cells. Furthermore, G-rich RNA sequence binding factor 1 (GRSF1) mediates the miR-346-dependent upregulation of hTERT by binding to the miR-346 middle sequence motif (CCGCAU) which forms a “bulge loop” when miR-346 is bound to the hTERT 3'UTR, facilitating the recruitment of hTERT mRNA to ribosomes to promote translation in an AGO2-independent manner. Conversely, miR-138 suppresses hTERT expression in an AGO2-dependent manner. Interestingly, replacement of the miR-138 middle sequence with that of miR-346 resulted in an upregulation of hTERT expression in a GRSF1-dependent manner. Moreover, miR-346 depends on GRSF1 to upregulate another target gene, activin A receptor, type IIB (ACVR2B), in which miR-346 “CCGCAU” motif is essential. These findings reveal novel mechanisms of miRNA-mediated upregulation of target gene expression and describe the coordinated action of multiple miRNAs to control the fate of a single target mRNA through binding to its 3'UTR.

Citation Format: Hua Tang. miR-346 and miR-138 competitively regulate hTERT in GRSF1- and AGO2-dependent manners, respectively. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr A12.
Abstract A21: MiR-106a and miR-106b affect growth and metastasis of lung adenocarcinoma
Authors: Enfield Katey SS , Rowbotham David A. , Holly Alice , Anderson Christine , Ng Kevin W. , Minatel Brenda de Carvalho , Dellaire Graham , Pastrello Chiara , Jurisica Igor , MacAulay Calum , Lam Stephen , Lam Wan L.
Published: May 2016

Introduction: MiR-106a and miR-106b are paralogs of the oncogenic miR-17~92, and have been associated with poor outcome and metastasis in several solid tumors. Their role in lung cancer is relatively unexplored. We characterized the expression of miR-106a and miR-106b in a clinical cohort of lung adenocarcinoma (AC) tumors and assessed their ability to regulate growth and metastasis in cell models.

Methods: MicroRNA (miRNA) expression was deduced from small RNA sequencing data derived from clinical lung AC specimens (60 localized, 27 with lymph node invasion) and paired non-malignant tissues. MiR-106a and miR-106b overexpression vectors and controls were stably transfected into immortalized non-malignant Human Bronchial Epithelial Cells (HBECs) and stage I AC cell lines with epithelial expression patterns by lentiviral delivery. Migration and invasion was assessed by Boyden chamber assay, while cell proliferation was assessed by BrdU incorporation assay. Expression of epithelial-to-mesenchymal transition (EMT) markers and other proteins of interest were assessed by Western Blot. Clinical associations in an external cohort were derived using publically available TCGA data.

Results: MiR-106a and miR-106b were significantly overexpressed in lung AC with lymph node invasion. Overexpression of miR-106a and miR-106b significantly increased proliferation of lung AC cell lines, and was associated with decreased levels of predicted target, p21. AC cell lines displayed a marked increase in metastatic phenotypes in vitro, and were associated with increased mesenchymal and decreased epithelial markers, characteristic of EMT. Importantly, tumors with high expression of both miR-106a and miR-106b and mesenchymal marker vimentin had significantly poorer outcome.

Conclusions: MiR-106a and miR-106b are overexpressed in metastatic lung AC. Lung AC cell models indicate these miRNAs are metastatic agonists, affecting the metastatic potential of cells at least in part via induction of EMT. A deeper characterization of this observation may reveal therapeutic intervention points, or, with the development of miRNA therapeutics, miR-106a/b may be promising targets to prevent or treat metastatic disease.

Citation Format: Katey SS Enfield, David A. Rowbotham, Alice Holly, Christine Anderson, Kevin W. Ng, Brenda de Carvalho Minatel, Graham Dellaire, Chiara Pastrello, Igor Jurisica, Calum MacAulay, Stephen Lam, Wan L. Lam. MiR-106a and miR-106b affect growth and metastasis of lung adenocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr A21.
Abstract A24: HDAC inhibition unmasks a microRNA-mediated mechanism of tumor suppression
Authors: Adams Clare M. , Hiebert Scott W. , Eischen Christine M.
Published: May 2016

Genetic lesions resulting in epigenetic dysregulation have a newly appreciated role in tumor initiation and progression. Alterations in epigenetic enzymes, such as histone deacetylases (HDACs), lead to dynamic changes in the epigenetic landscape that are postulated to alter the expression of critical mediators of tumorigenesis. HDACs not only alter the expression of protein-coding genes, but also affect noncoding RNAs, including microRNA (miRNA). Recent evidence suggests that miRNA are one of the largest classes of regulatory molecules and, given their broad impact on gene expression, it is not surprising they have a significant role in tumorigenesis. Although, small molecule inhibitors of HDACs have demonstrated therapeutic utility, the molecular events underlying the observed selective killing of cancer cells are incompletely resolved. We determined that HDAC inhibition in numerous hematopoietic malignancies relieved the Myc-mediated transcriptional repression of miRNA with known tumor suppressive properties, namely the miR-15 and let-7 families. Specifically, following HDAC inhibition, Myc, which normally represses the miR-15 and let-7 families in cancer cells, transcriptionally activated their expression. In addition, we demonstrated that Myc was required for this HDAC inhibition-induced miRNA upregulation. As a result, transcript levels of the miR-15 and let-7 families increased, which subsequently targeted and decreased the expression of anti-apoptotic Bcl-2 and Bcl-xL, respectively, inducing apoptosis in multiple hematopoietic malignancies. Unexpectedly, we demonstrated that, in normal cells, dysregulated Myc transcriptionally upregulated these miRNA as a means to prevent cellular transformation, revealing a novel mechanism whereby Myc induces apoptosis independent of the p53 tumor suppressor. Our results expose a previously unknown, and surprisingly general, mechanism of Myc-induced apoptosis that capitalizes on miRNA. Importantly, our data provide insight into the molecular underpinnings following HDAC inhibition and contribute new knowledge that should aid in the development of improved cancer therapeutics.

Citation Format: Clare M. Adams, Scott W. Hiebert, Christine M. Eischen. HDAC inhibition unmasks a microRNA-mediated mechanism of tumor suppression. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr A24.
Abstract A32: The RNA-binding protein DDX1 promotes primary microRNA maturation and inhibits ovarian tumor progression
Authors: Han Cecil , Lu Xiongbin , Zhang Xinna
Published: May 2016

MicroRNAs (miRNAs) are small endogenous non-coding RNAs that regulate gene expression by repressing translation and/or promoting degradation of their target mRNAs. Aberrant dysregulation of mature miRNAs is commonly observed in many human cancers and can have a causative function in tumorigenesis. In the nucleus, primary miRNAs (pri-miRNAs) are first processed by the microprocessor containing RNaseIII enzyme Drosha and its cofactor DGCR8. Although the efficiency of Drosha-mediated process is crucial for determining miRNA abundance, the mechanisms for dynamic and specific regulation of miRNA biogenesis in the Drosha microprocessor are poorly understood. A better understanding of miRNA regulation is required to determine how aberrant miRNA activity mechanistically contributes to tumorigenesis and to predict therapeutic target miRNAs. Increasing evidence has shown that regulatory RNA-binding proteins may confer the specificity on the core components (Drosha and DGCR8) in the Microprocessor for recruiting and processing premature pri-miRNAs.

Here, we identified a RNA-binding protein, DEAD-box RNA helicase 1 (DDX1) as a key regulatory component in the Drosha complex, which promotes the processing of a subset of miRNAs including miR-200 family (miR-200a, -200b, -200c, -141, -429). A suppressive role of miR-200s in cancer metastasis has been well known in several cancer types such as ovarian, kidney renal and lung cancer. Inhibiting DDX1 promotes tumor burden in the previous syngeneic ovarian mouse models. Consistent with, low level of DDX1 is associated with poor clinical outcome in patients with serous ovarian cancer and kidney renal clear cell carcinoma. Interestingly, DDX1-dependent miR-200a and miR-200b were also induced after DNA damage and this induction is facilitated by the ATM-mediated phosphorylation of DDX1. These findings suggest that DDX1 may be a key modulator in miRNA maturation in response to cellular signaling pathway and that alteration of level/function of DDX1 may play an important role in tumor progression through dysregulated microRNA pathway.

Citation Format: Cecil Han, Xiongbin Lu, Xinna Zhang. The RNA-binding protein DDX1 promotes primary microRNA maturation and inhibits ovarian tumor progression. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr A32.