bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4421: IGF2 regulates mitochondrial cell energy phenotype and biogenesis in TNBC cells
Authors: Pagan Maria L. , Radhakrishnan Vinodh Kumar , Leon Daisy De
Published: July 2017

Triple-negative breast cancer (TNBC) is very aggressive, resistant to chemotherapy and more likely to relapse, causing the worst prognosis. African American (AA) women suffer higher incidence and mortality of TNBC due to the expression of high levels of Insulin Growth Factor 2 (IGF2) which promotes tumor progression, metastasis, and chemoresistance. Also, it has been established that functional mitochondria and mitochondrial DNA (mtDNA) are essential for cancer cell growth. Mutations and/or reductions in mtDNA copy number that alter the Oxidative Phosphorylation (OXPHOS) physiology are common features of TNBC. We have demonstrated that mtDNA content is lower in CRL-2335 AA TNBC cell line when compared to the CRL-2335 IGF2 knockout cell line. Thus, we propose that IGF2 regulates the mtDNA content. This study was designed to demonstrate if IGF2 regulates mitochondrial genes to determine the cell energy phenotype. An XFp analyzer was used to study the mitochondrial function in terms of OCR (Oxygen Consumption Rate/Mitochondrial Respiration) and ECAR (Extracellular Acidification Rate/ Glycolysis) in the wild type and IGF2 stable knockout of CRL-2335 AA TNBC cells. Real Time PCR was performed to study the gene expression pattern of IGF2, PGC1α and PGC1β. PGC1α and PGC1β are critical genes in the regulation of the mitochondrial biogenesis, thus, they are important in the cellular metabolic phenotype. Utilizing the Seahorse metabolic system we assessed cell energy phenotype and alterations in terms of mitochondrial respiration rate (OCR) % and Glycolysis (ECAR) %. Our preliminary results showed that the overall OCR and ECAR of the stressed CRL-2335 AA TNBC cells was altered according to the levels of IGF-II expressed. Furthermore, the results demonstrated that there was a metabolic shift in the CRL-2335 AA TNBC cells towards the glycolytic pathway when IGF2 was knockout in comparison to the wild type CRL-2335 cells. Also, IGF2 knockout cells showed higher gene expression rate of PGC1β as compared to the wild type. The above data confirms that IGF2 plays a critical role in determining the cell energy phenotype.

Citation Format: Maria L. Pagan, Vinodh Kumar Radhakrishnan, Daisy De Leon. IGF2 regulates mitochondrial cell energy phenotype and biogenesis in TNBC cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4421. doi:10.1158/1538-7445.AM2017-4421
Abstract 4425: Analysis of host-Epstein-Barr virus microRNA expression in nasopharyngeal carcinoma
Authors: Su Wen-Hui , Zhang Jia-Hao , Chang Kai-Ping
Published: July 2017

In the tumorigenesis process, miRNAs could act as oncogenes or tumor suppressor genes in the cancer cells by regulating miRNA target genes. Nasopharyngeal carcinoma (NPC) associated Epstein-Barr virus (EBV) can also influence the occurrence of NPC through its miRNAs. In this study, we used the next generation sequencing technology to analysis host-viral miRNA profiles in 24 NPC and 5 adjacent normal tissues. In average, 10.62% of mappable reads mapped to EBV genome in NPC tissue. Most of the small RNAs in NPC tissues which mapped to EBV genome were miRNAs (95.51%), rest of them were EBERs (1.80%) and v-snoRNA1 (2.68%). The percentages of EBV miRNA in total human and EBV mature miRNA reads were ranged from 4% to 51% (average 29.61%). For EBV miRNAs, the expression level of BART10-3p and BART9-3p were the top two in NPC tumors. BART20-5p, BART19-3p and BART21-5p were correlated to tumor stage. On the other hand, there were 11 and 34 human miRNAs differentially up- and down-regulated in NPC tissue, respectively. The present research provides the first evidence obtained through direct sequencing to systematically and comprehensively describe the expression of EBV and human miRNAs in NPC tissue.

Citation Format: Wen-Hui Su, Jia-Hao Zhang, Kai-Ping Chang. Analysis of host-Epstein-Barr virus microRNA expression in nasopharyngeal carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4425. doi:10.1158/1538-7445.AM2017-4425
Abstract 443: Lymphatic endothelium protects breast cancer cells from death by inducing metabolic adaptations
Authors: Berisa Mirela , Podgrabinska Simona , Nicolay Brandon , Mostoslavsky Raul , Chipuk Jerry , Skobe Mihaela
Published: July 2017

The lymphatic vasculature is an important pathway for breast cancer dissemination, yet it is not understood whether and how the lymphatic vessel microenvironment influences cancer metastasis. We demonstrate that lymphatic endothelial cells (LECs) promote survival of triple-negative breast cancer cells (TNBCs) under stress by improving mitochondrial function and inducing metabolic shift to promote cellular energy production. LECs protected TNBCs from death in vitro induced by the loss of attachment and nutrient deprivation. Cell death was preceded with a sharp increase in reactive oxygen species (ROS), strong up-regulation of Nrf2-mediated oxidative stress response genes and a rapid decline of mitochondrial activity. LECs lowered ROS levels, decreased mitochondrial superoxide formation and enhanced mitochondrial activity in TNBCs. RNAseq transcriptome analysis identified key regulator of mitochondrial metabolism and cellular bioenergetics, peroxisome proliferator-activated receptor gamma coactivator (PPARGC1A/PGC-1α, to be specifically up-regulated in breast cancer cells by LEC-derived factors. Inhibition studies demonstrated that the TNBC survival was dependent on pentose phosphate pathway (PPP) activity. Notably, LECs induced a metabolic shift from glycolysis to fatty-acid oxidation (FAO) and oxidative phosphorylation to maintain ATP and sustain cell viability. These data demonstrate that lymphatic endothelium promotes survival of breast cancer cells by regulating energy production and maintaining redox homeostasis. Our findings suggest that lymphatic endothelium may facilitate metastasis by promoting survival of breast cancer cells within the lymphatic vasculature.

Citation Format: Mirela Berisa, Simona Podgrabinska, Brandon Nicolay, Raul Mostoslavsky, Jerry Chipuk, Mihaela Skobe. Lymphatic endothelium protects breast cancer cells from death by inducing metabolic adaptations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 443. doi:10.1158/1538-7445.AM2017-443
Abstract 4447: Analysis of small RNA-seq data for differential expression of small noncoding RNAs in human colorectal cancer
Authors: Koduru Srinivas V. , Tiwari Amit K. , Hazard Sprague W. , Mahajan Milind K. , Ravnic Dino J.
Published: July 2017

Background: Cancer is a major cause of deaths worldwide, despite improved healthcare and technologies. Recent advancements in genome sequencing have led to the rapid study of the whole transcriptome and small RNAs with their biological functions. Non-coding RNAs (ncRNAs) play an important role in biological processes that greatly impact biomarker development for diagnosis, prognosis, and therapy. Non-coding RNAs such as microRNAs (miRNA), Piwi-interacting-RNAs (piRNAs), small nuclear/nucleolar RNAs (sn/snoRNAs) have recently been studied to understand their biology and pathology.

Results: In the present study, we used eight matched colorectal patient tissue samples (benign, tumor, and metastasis) small RNA sequencing data remapped for various small RNA annotation. We identified aberrant expression of 13 miRs in tumor and metastasis specimens [tumor vs benign group (19 miRs) and metastasis vs benign group (38 miRs)] of which five were upregulated, and eight were downregulated, during disease progression. We also investigated pathway analysis on abbarent expression of miRNAs, which showed majority of miRs involved in the colon other types of cancers. Further analysis of piRNAs revealed that six piRNAs in tumor vs benign and 24 in metastasis vs benign samples (commonly in both groups, only two piRNAs). Additionally, we examined other types of small RNAs [sn/snoRNAs, mt_rRNA, misc_RNA, nonsense mediated decay (NMD) and rRNA], we identified 15 in tumor vs benign and 104 metastasis vs benign and only four in commonly expressed.

Conclusion: In summary, our results identified multiple sncRNAs during colorectal cancer progression which needs to be further validated and can be used for prognosis, diagnosis, and therapeutic potentials.

Citation Format: Srinivas V. Koduru, Amit K. Tiwari, Sprague W. Hazard, Milind K. Mahajan, Dino J. Ravnic. Analysis of small RNA-seq data for differential expression of small noncoding RNAs in human colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4447. doi:10.1158/1538-7445.AM2017-4447
Abstract 4466: The key role of Abi1 loss in dysregulating cell-cell adhesion during prostate cancer tumorigenesis
Authors: Das Disharee , Hryniewicz-Jankowska Anita , Hehnly Heidi , Post Dawn , Bratslavsky Gennady , Kotula Leszek
Published: July 2017

Prostate cancer accounts for 20-25% of cancer cases every year and is predicted at over 180,000 new cases in 2016. However, the mechanisms of prostate cancer initiation and castration resistance still remains poorly understood. Our previous research shows mutations and deletions in Abelson interactor 1 (Abi1), an integral part of WAVE complex, in prostate tumor samples. Moreover, WAVE complex levels are inversely correlated to prostate cancer recurrence and castration resistance, outlining their clinical significance. A common phenomenon found early on in most cancers, including prostate cancer, is dysregulated cell-cell adhesion. This points towards a possible tumor suppressor mechanism, since Abi1 and the WAVE complex are in fact the primary regulators of branched actin polymerization via the Arp2/3 complex, and are critical for the formation and maintenance of cell-cell adhesion complexes. Hence, to study Abi1 in prostate cancer, we generated a prostate-specific conditional Abi1 knock-out (KO) mouse. In these mice, we observed evidence of prostatic intraepithelial neoplasia (PIN), a precursor of invasive carcinoma, with changes in proliferation and cell-cell adhesion markers. The goal of this study is to elucidate the mechanism by which the loss of Abi1 leads to the development of PIN in these mice. We are leading this analysis by primary 3D cultures with cells from our Abi1 KO prostates, and 2D cultures with mouse embryonic fibroblasts (MEFs). Loss of Abi1 leads to increase in size of the cultured spheroids, which indicates increased proliferation and dysregulated cell-cell adhesion. Also, adherens junction (AJ) markers E-cadherin and β-catenin levels are lower in western blots and immunostaining, suggesting defects in cell-cell adhesion. In MEFs, we observe loss of membrane localization of AJ proteins in Abi1 KO cells. Loss of Abi1 is often associated with upregulation of Abi2, which may be a contributing factor to the phenotype. To validate our findings in a human system, we have recently developed a CRISPR-mediated KO of Abi1 in the RWPE-1 non-tumor prostate cell line. We will use this KO cell line to determine cell-cell adhesion formation and maintenance in 2D and 3D cultures. In conclusion, loss of Abi1 leads to dysregulation of cell-cell adhesion in prostate epithelial cells, which may be a key mechanism for tumor development and progression in prostate cancer. Supported by NCI R01 CA161018 (LK).

Citation Format: Disharee Das, Anita Hryniewicz-Jankowska, Heidi Hehnly, Dawn Post, Gennady Bratslavsky, Leszek Kotula. The key role of Abi1 loss in dysregulating cell-cell adhesion during prostate cancer tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4466. doi:10.1158/1538-7445.AM2017-4466
Abstract 4477: HnRNP Q1 translationally increases the expression of Aurora-A and promotes tumorigenesis in colorectal cancer
Authors: Hung Liang-Yi
Published: July 2017

Abnormally elevated Aurora-A can cause mitotic defects and aneuploidy, which might lead to genomic instability and tumorigenesis. According to the literatures, overexpressed Aurora-A may result from gene amplification, RNA transcriptional activation, or increased protein stability. Our previous study has demonstrated that Aurora-A can be translational upregulated in colorectal cancer. By biotin pull-down assay and in vivo translational assay, we found that Aurora-A mRNA can be translational upregulated by heterogeneous nuclear ribonucleoprotein (hnRNP) Q1, a RNA binding protein. Hn RNP Q1 has been implicated in many posttranscriptional regulatory processes including mRNA splicing, RNA metabolism and translation. In this study, we further clarified the regulatory mechanism of hnRNP Q1 in translational regulating Aurora-A mRNA, as well as investigated the clinical role of hnRNP Q1 in colorectal carcinogenesis. Our results indicated that hnRNP Q1 can bind to the Aurora-A mRNA 5’-untranslated region (5’-UTR). Ribosomal protein S6-IP assay further indicated that hnRNP Q1 can enhance the translational efficacy of Aurora-A mRNA. Ribosomal profiling assay further confirmed the translational regulation of Aurora-A mRNA by hnRNP Q1. Overexpression of hnRNP Q1 promotes cell proliferation and tumor growth. The expression level of hnRNP Q1 is positively correlated with Aurora-A in colorectal cancer. Taken together, our data indicate hnRNP Q1 is a novel trans-acting factor that binds to Aurora-A mRNA 5’-UTRs and regulates its translation, which increases cell proliferation and contributes to tumorigenesis in colorectal cancer.

Citation Format: Liang-Yi Hung. HnRNP Q1 translationally increases the expression of Aurora-A and promotes tumorigenesis in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4477. doi:10.1158/1538-7445.AM2017-4477
Abstract 4479: Cancer cell ribogenesis: MYC and the integrity of the RNA polymerase I-rRNA machinery
Authors: Rossetti Stefano , Wierzbicki Andrzej J. , Sacchi Nicoletta
Published: July 2017

Upregulation of RNA Polymerase (Pol I)-mediated transcription of ribosomal RNA (rRNA) and increased ribogenesis, which are necessary to sustain the increased metabolic demand of highly proliferating cancer cells, are hallmarks of cancer. Increased rRNA transcription can be due deregulation of tumor suppressors and oncogenes that affect Pol I activity. Overexpression of the MYC oncogene, a potent Pol I activators, is particularly frequent in cancer. In addition, based on our analysis of the Cancer Genome Atlas (TCGA), amplification/upregulation of genes encoding for basal components of the Pol I transcriptional machinery is also frequent in cancers of various histotype (Rossetti et al., Cell Cycle, 2016). By using breast and ovarian epithelial cells, we mechanistically found that: 1) ectopic expression of either MYC or RRN3 (TIF-IA), a key Pol I basal component, by increasing rRNA synthesis, is sufficient to induce in vitro transformation phenotypes and to promote cell proliferation; 2) there is a causal link between MYC overexpression and RRN3 upregulation; 3) MYC- or RRN3-induced rRNA upregulation sensitizes cells to the anti-proliferative action of drugs inhibiting rRNA transcription. Our findings provide a rationale for using drugs targeting rRNA transcription to curb proliferation of cancers cells due to rRNA upregulation by MYC.

Funding for this study was provided by an RPCI-UPCI Ovarian Cancer Spore DRP award (NS), the NCI R01 CA127614 grant (NS), the Terri Brodeur Breast Cancer Foundation (SR), the Susan Komen Foundation (SR), and the NCI P30 CA016056 institutional grant.

Citation Format: Stefano Rossetti, Andrzej J. Wierzbicki, Nicoletta Sacchi. Cancer cell ribogenesis: MYC and the integrity of the RNA polymerase I-rRNA machinery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4479. doi:10.1158/1538-7445.AM2017-4479
Abstract 4487: A critical role of PARylation in regulating the functions of OVOL2
Authors: Zhang Rui , Li Boan , Liou Yih-Cherng
Published: July 2017

Poly(ADP-ribosyl)ation (PARylation) is one of the post-translational modifications, by which the ADP-ribose (PAR) polymers are added to the specific protein by a PAR polymerase (PARP). In the about 50 years' studies, this specific modification has been verified to be related to multiple important biological functions including cell death, cell differentiation, histone modification, transcriptional regulation, DNA repair and genome stability. Ovo-like 2 (OVOL2), a novel zinc finger transcriptional factor, contains a Snail/Gli (SNAG) motif at the N-terminal to possess a transcriptional regulatory activity and four DNA-binding Cys2-His2 (C2H2) zinc finger domains at the C-terminal to bind with DNA. OVOL2 is demonstrated to be involved in the cranial neural tube, angiogenesis, heart and placental development. In addition, OVOL2 can also suppress cell cycle and terminate differentiation of keratinocyte by directly inhibiting of c-Myc and Notch1. However, the regulation and biological function of OVOL2 are still largely unknown. In this study, we identify the binding targets, PARP1 and NPM1, of OVOL2. In addition, we report that the function of OVOL2 is regulated by PARP1 through PARylation, which modifies on C2H2 zinc finger domains and plays an important role in the regulation of OVOL2 functions. PARylated OVOL2, but not the 3K mutant, alters focal adhesion and cell cycle, resulting in the formation of polyploidy in cells. Our findings not only have significant implications for the cellular functions of OVOL2, but also provide a better understanding on the multiple biological functions of the PARylation.

Citation Format: Rui Zhang, Boan Li, Yih-Cherng Liou. A critical role of PARylation in regulating the functions of OVOL2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4487. doi:10.1158/1538-7445.AM2017-4487
Abstract 449: Reduced argininosuccinate synthetase expression in refractory sarcomas: impacts on therapeutic potential and drug resistance
Authors: Kobayashi Eisuke , Kim Youngji , Kubota Daisuke , Suehara Yoshiyuki , Kawai Akira , Kitano Shigehisa
Published: July 2017

Introduction: Treating drug-resistant sarcomas remain a major challenge. The present study aimed to identify a novel therapy for drug-resistant sarcomas based on a metabolic errors involving argininosuccinate synthetase1 (ASS1).

Methods: We assessed the expression of ASS1 and P-glycoprotein (P-gp) in osteosarcoma (KHOS), doxorubicin (Dox)-resistant osteosarcoma (KHOSR2), epithelioid sarcomas (ES-X and VAESBJ), alveolar soft part sarcoma (ASPS-KY), and each clinical specimen. Each cell was cultured in arginine-containing and arginine-free media. Cell growth was assessed using an XTT assay and flow cytometry. We analyzed the induction of autophagy in arginine-free medium. Moreover, we assessed the expression of P-gp in after suppressing ASS1 in Dox-sensitive cells (MCF-7, KHOS) and in after transfecting the ASS1 into Dox-resistant cells (ES-X, VAESBJ, ASPS-KY and KHOSR2).

Results: The expression of ASS1 was reduced in Dox-resistant sarcoma cells. Immunohistochemistry (IHC) and real-time PCR showed that there was interestingly an inverse relationship between the expression of ASS1 and the expression of P-gp. The inhibition of cellular proliferation with G1-arrest was shown to lead to autophagy with arginine deprivation. In addition, the combination of autophagy inhibitor plus arginine deprivation was more effective than arginine deprivation alone. In cells in which the expression of ASS1 was suppressed, the expression of P-gp was upregulated in comparison to negative controls.

Discussion: These results indicate that the reduced expression of ASS1 expression in Dox-resistant sarcomas may contribute to drug resistance. ASS1 deficiency is a potential target for novel drug therapies. The combination of arginine deprivation therapy and an autophagy inhibitor may have anti-tumor effects in refractory sarcomas.

Significance: As the induction of autophagy by the deprivation arginine may have a pro-survival role in patients with ASS1-deficient sarcomas, the combination of arginine deprivation therapy with autophagy modulators might potentiate anti-tumor effects in patients with drug-resistant sarcomas.

Citation Format: Eisuke Kobayashi, Youngji Kim, Daisuke Kubota, Yoshiyuki Suehara, Akira Kawai, Shigehisa Kitano. Reduced argininosuccinate synthetase expression in refractory sarcomas: impacts on therapeutic potential and drug resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 449. doi:10.1158/1538-7445.AM2017-449
Abstract 4497: Single-cell protein-mRNA co-detection to reveals subpopulation-specific correlation patterns
Authors: Gong Haibiao , Wang Xiaohui , Liu Benjamin , Holcomb Ilona , Dakshinamoorthy Gajalakshmi , Ooi Aik , Sun Gang , Ramakrishnan Ramesh
Published: July 2017

We have developed a streamlined workflow for multiplexed co-detection of proteins and mRNAs in single reactions at single-cell resolution. This workflow couples OER for protein detection with reverse transcription (RT) for mRNA detection. In this study, we examined 31 overlapping targets, from a panel of 84 protein and 40 mRNA assays, across three different cell lines (A549, SKBR3 and K562). Unsupervised gene expression profiling analysis, including principal component analysis and hierarchical clustering revealed different aspects of the protein-mRNA relationship. The top 10 genes contributing to cell differentiation were identified for both proteins and mRNAs, and eight of them overlap. This result implies an overall concordance of certain protein and mRNA expression in different cell lines. Our results also demonstrate that cells can be classified into subpopulations based on protein-mRNA expression profiles, and different subpopulations have distinct correlation coefficient values. The correlation between CCNB1 protein and mRNA was low (R = 0.2884) in the whole population. However, there was a subpopulation in which CCNB1 protein correlated strongly with its mRNA. When this subpopulation was analyzed, the R value was 0.8350, while therest of the cells had an R value of 0.0233. More interestingly, in subpopulation I CCNB1 protein level correlated positively with CCNA2 protein (R = 0.9102) but negatively with CCNE1 protein(R = - 0.5249). The observed correlation patterns of these genes match their known expression profiling at cell-cycle phases of S, G2 and M, suggesting that this subpopulation may represent actively dividing cells. This study further indicated that systematic comparisons of protein and mRNA expression data in single cells can reveal additional layers of complexity which are not attainable at bulk levels.

Citation Format: Haibiao Gong, Xiaohui Wang, Benjamin Liu, Ilona Holcomb, Gajalakshmi Dakshinamoorthy, Aik Ooi, Gang Sun, Ramesh Ramakrishnan. Single-cell protein-mRNA co-detection to reveals subpopulation-specific correlation patterns [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4497. doi:10.1158/1538-7445.AM2017-4497
Abstract 4517: A CRISPR approach to monitoring hypoxia-inducible proteins in real-time
Authors: Schwinn Marie K. , Machleidt Thomas , Binkowski Brock F. , Eggers Christopher T. , Wood Keith V.
Published: July 2017

Hypoxia-inducible factor 1A (HIF1A) regulates expression of genes implicated in various aspects of oncogenesis, including angiogenesis, cell survival, metastasis, and glucose metabolism. Overexpression or hypoxia-induced stabilization of HIF1A has been associated with poor prognosis in cancer patients, making HIF1A and its associated pathway a high-profile target for anticancer therapies. We sought to develop a live-cell assay to monitor abundance of endogenous HIF1A and HIF1A-inducible proteins that could be used to identify potent and specific inhibitors of the hypoxia signaling pathway. To accomplish this goal, mammalian cell lines were edited by CRISPR using a Cas9:crRNA ribonucleoprotein complex with a single-stranded oligonucleotide donor DNA to introduce the HiBiT tag at the C-terminus of HIF1A and a number of known hypoxia-inducible proteins, including BNIP3, ANKRD37, HILDPA and KLF10. The 11 amino acid HiBiT peptide and its complementing 18 kDa polypeptide, known as LgBiT, spontaneously reconstitute into an active luciferase derived from the NanoLuc enzyme. Co-expression of LgBiT in edited cells, followed by addition of the cell-permeable luciferase substrate, leads to generation of a bright, steady luminescent signal that directly correlates with abundance of the HiBiT fusion. The edited cells were treated with several known modulators of the HIF1A signaling pathway, and changes in the abundance of the protein fusions were followed in real-time by monitoring luminescence. The HiBiT tag was also used to validate size and subcellular localization of the fusion proteins using bioluminescence imaging and antibody-free blotting. As expected, all tested compounds induced HIF1A accumulation. However, the downstream targets of HIF1A generated differing response to the chemical modulators, warranting further investigation into the modes by which these compounds act. By coupling the speed and efficiency of CRISPR-mediated editing with the small size and brightness of HiBiT, it was possible to generate a live-cell assay to monitor abundance of proteins along the HIF1A pathway. This assay could easily be adapted to screen for compound-induced effects on protein levels of HIF1A, as well as HIF1A- induced changes in expression patterns.

Citation Format: Marie K. Schwinn, Thomas Machleidt, Brock F. Binkowski, Christopher T. Eggers, Keith V. Wood. A CRISPR approach to monitoring hypoxia-inducible proteins in real-time [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4517. doi:10.1158/1538-7445.AM2017-4517
Abstract 4520: Hepatitis B virus X protein regulates hypoxia-inducible factor-1alpha (HIF-1 alpha) and lysyl oxidase like 2 (LOXL2) pathway in hepatocellular carcinoma
Authors: Chan Cerise Yuen-Ki , Tse Aki Pui-Wah , Chiu Elley Yong-Tuen , Sze Karen Man-Fong , Koh Hui-Yu , Ng Irene Oi-Lin , Wong Carmen Chak-Lui
Published: July 2017

Hepatocellular carcinoma (HCC), the most common form of primary liver cancer, is a fatal malignancy and is prevalent in HBV-endemic geographical areas. Hepatitis B virus (HBV) infection is a major etiologic factor of HCC. HBV is a double-stranded DNA virus and encodes for a viral onco-protein called transactivator protein X (HBx) which interacts with the host proteins to enhance proliferative potential of the host cells. Here, using Tet-ON and Tet-OFF HBx inducible systems in HBV-negative HCC cell lines, we showed that induced expression of HBx stabilizes hypoxia-inducible factor-1α (HIF-1α) protein under hypoxia. Reversely, knockdown of HBx in HBV-positive HCC cell lines reduced HIF-1α protein stabilization under hypoxia. More intriguingly, we found that induced HBx expression up-regulated the mRNA and protein expression a known HIF-1α transcriptional target, lysyl oxidase like 2 (LOXL2) in HCC. Our previous study showed that LOXL2 cross-linked collagen in the extracellular matrix to promote HCC metastasis. Scanning electron microscopy demonstrated that knockdown of HBx in HCC cell lines markedly reduced formation of collagen fibers in vitro. Transwell invasion assay showed that knockdown of HBx reduced HCC invasive ability. Picrosirius red staining further showed that knockdown of HBx reduced collagen cross-linking in vivo and repressed HCC growth and metastasis. Taken together, our study unprecedentedly show the HBx remodels the ECM through HIF-1α/LOXL2 pathway to promote HCC metastasis.

Citation Format: Cerise Yuen-Ki Chan, Aki Pui-Wah Tse, Elley Yong-Tuen Chiu, Karen Man-Fong Sze, Hui-Yu Koh, Irene Oi-Lin Ng, Carmen Chak-Lui Wong. Hepatitis B virus X protein regulates hypoxia-inducible factor-1alpha (HIF-1 alpha) and lysyl oxidase like 2 (LOXL2) pathway in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4520. doi:10.1158/1538-7445.AM2017-4520
Abstract 4541: Understanding the dose and schedule dependence of efficacy for the Wee1 inhibitor AZD1775 in xenograft and patient derived explant models by mathematical modelling
Authors: Yates James W. , Cadogan Elaine , Hare Jennifer , Hughes Adina , Polanska Urszula M. , O'Connor Mark , Critchlow Susan E.
Published: July 2017

AZD1775 is a highly selective, small-molecule inhibitor of WEE1 being developed to treat patients with advanced solid tumors, as monotherapy and in combination with olaparib.

Experimental Procedures Patient-derived explant (PDX) and xenografted models with a range of sensitivities to AZD1775 were tested for response to different doses and schedules of AZD1775. WEE1 inhibition was assessed by pCDK1 as a direct substrate of Wee1 and RRM2 as a surrogate of CDK2 activity. Concentration of AZD1775 in plasma was measured in the same animals. Mathematical models were developed to describe the relationship between drug exposure, biomarker modulation and resulting tumour growth inhibition.

Results The PKPD relationship of both pCDK1 and RRM2 could be described by the mathematical model with significant delay between PK and RRM2 reduction due to this being a protein degradation event. Three factors influenced anti-tumour activity in the A427 xenograft and three TNBC PDX models, namely the AZD1775 dose, the number of consecutive days of dosing and the number of days between AZD1775 doses. The minimally effective preclinical dose with anti-tumour activity was 60 mg/kg od. TGI was seen to increase with increased days of consecutive AZD1775 dosing from 3 days on, such that a 5 days on/9 days off schedule was more efficacious than 3 days on/4 days off schedule for two weeks. A mathematical model successfully described this efficacy by using the predicted dose and schedule dependent reduction in pCDK1.

Conclusions The insights from this predictive modelling informed the starting dose of 125mg BID AZD1775 on a 5 day on/9 day off schedule; with the clinical goal to optimize the days of consecutive AZD1775 dosing as well as the maximum tolerated dose.

Citation Format: James W. Yates, Elaine Cadogan, Jennifer Hare, Adina Hughes, Urszula M. Polanska, Mark O'Connor, Susan E. Critchlow. Understanding the dose and schedule dependence of efficacy for the Wee1 inhibitor AZD1775 in xenograft and patient derived explant models by mathematical modelling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4541. doi:10.1158/1538-7445.AM2017-4541
Abstract 4546: Structural evaluation of mutations identified on Cue domain of FANCD2
Authors: Siddiqui Mohd Quadir , Varma Ashok K.
Published: July 2017

Fanconi anemia complementation group D2 (FANCD2) protein plays pivotal role in DNA interstrand crosslink (ICL) repair and genome stability. FANCD2 comprises conserved CUE domain at N terminus, which is important for DNA ICL repair and protein stability. However, molecular mechanism associated to genetic alteration has not well characterized. Here, we have carried out multimodal approaches to explore folding pattern of FANCD2 Cue domain and functionally relevant mutations. Dynamic Light Scattering and Mass Spectrometry data of purified proteins could revealed that wild- type protein is predominantly monomeric, but mutated counterparts are exhibiting oligomeric properties. Secondary, tertiary structure assessment and their thermal stability of wild- type, mutants were studied by Circular Dichroism (CD) and Fluorescence spectroscopy. Furthermore, binding affinity and kinetics of Cue domain and mutants with monoubiquitin was evaluated by surface plasmon resonance (SPR). Molecular modelling, simulation, normal mode analysis, principal component analysis and docking studies were carried out to understand in-depth conformational dynamics and interactions. References:- 1. Regulation of the Fanconi anemia pathway by a CUE ubiquitin-binding domain in the FANCD2 protein. Rego, M. A. et al, Blood 120: 2109-2117,(2012). 2. Hypomorphic mutations in the gene encoding a key Fanconi anemia protein, FANCD2, sustain a significant group of FA-D2 patients with severe phenotype. Kalb et al, Am J Hum Genet 80: 895-910, (2007)

Citation Format: Mohd Quadir Siddiqui, Ashok K. Varma. Structural evaluation of mutations identified on Cue domain of FANCD2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4546. doi:10.1158/1538-7445.AM2017-4546
Abstract 4552: ssCOPA: a novel method with improved sensitivity to assess statistical significance for cancer outlier profile analysis of gene expression
Authors: Gao Hong , Ng Sam , Gierman Hinco J.
Published: July 2017

To understand the genetic mechanism of disease, finding biomarkers and drug targets are a major focus of academic and pharmaceutical research. However, it has become clear that many important genes driving diseases like cancer, are deregulated in a relatively small group of outliers, e.g. amplified and fusion genes. Cancer outlier profile analysis (COPA), introduced by Tomlins et al, has raised interest in detecting genes with outlier expression within a group, particularly in cancer patients. However, the magnitudes of the COPA scores are not directly comparable across different studies or with different input parameters. Also, integrating COPA scores across multiple studies in a meta-analysis is problematic. Current solutions to performing meta-analyses, including rank-based methods and binomial test, require large study numbers without adjustment for the differences in study size. Also, within a single study COPA scores alone do not provide a P-value, and it is impossible to know which outlier genes are statistically significant. Here we developed three novel methods to address these problems by computing a significance for COPA scores, including Bootstrap resampling, Bootstrap + generalized Pareto distribution (GPD) and Binomial method for statistical significance of COPA (ssCOPA), to enable the comparison of COPA score across studies or parameters. We first evaluated our methods using two breast cancer studies. The P-values generated by Binomial method match closely to Bootstrap resampling using 10,000 randomizations, better than Bootstrap + GPD. In addition, the Binomial method requires significantly less computing time than the other methods. We further validated our methods with a meta-analysis of five breast cancer studies and compared the results with the top-ranking genes of a large-scale meta-analysis of 31 breast cancer studies by ONCOMINE. We show that our P-value-based approaches (P = 10-17~10-24) achieves significantly better concordance than the Binomial test (P = 10-14). Data and methods are available for use on the Illumina BaseSpace® CohortAnalyzer platform (formerly known as NextBio Clinical).

Citation Format: Hong Gao, Sam Ng, Hinco J. Gierman. ssCOPA: a novel method with improved sensitivity to assess statistical significance for cancer outlier profile analysis of gene expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4552. doi:10.1158/1538-7445.AM2017-4552
Abstract 4554A: Simple, efficient, and quantitative approach for determination of synergism, additive effect, and antagonism of drugs in vivo using combination index method: a proposition for clinical protocol design and regulatory synergy claims
Authors: Fu Jianing , Chou Ting-Chao
Published: July 2017

Based on the systematic dynamic rate derivations with the mass-action principle and mathematical induction-deduction, the median-effect equation of Chou, and the combination index (CI) equation and their algorithm allow quantitative determination of synergism (CI<1), additive effect (CI=1) and antagonism (CI>1) (Pharmacol Rev 58:621-681, 2006). Based on Google Scholar Citations, the CI simulation has been cited 7,712 times in >800 bio-medical journals as of 10.19.2016. However, most citing papers were with experiments in vitro, relative few in vivo and rarely in clinical trials. This is despite the fact that the CI method equation, algorithm, and the CompuSyn software are the same for in vitro and in vivo, although the practical aspects such as sample size, time and cost, the readiness of the end-point measurement, and the ethical considerations are different. Our recent publication (in Fu J et al. Synergy 3: 15-30, 2016) of two-drug combination (docetaxel and T-607 compound, 1:2) against HCT-116 colon carcinoma xenograft in nude mice has demonstrated very desirable synergy. We now illustrate the experimental and data-analytical details of the experimental planning and design, execution, measurements, for docetaxel and T-Compound combination against MX-1 mammary carcinoma xenograft in nude mice. Treatment Q3Dx4 i.v. starting Day 7, and the tumor sizes and bodyweight changes measured on Days 16, 19, and 22. A total of 50 nude mice were used (each drug and combinations, 3 doses each (n=5) plus a control group of 5). Full spectrum of synergism (CI=0.79 to 0.93) were observed on Days 19 and 22. Automated simulation were carried out for dose-effect curve, ME plot, Fa-CI plot, isobologram, and Fa-DRI (Dose-Reduction Index) plot with CompuSyn software (Free download available upon registration via www.combosyn.com). This simple, efficient, “econo-green” bio-research paves the way for drug combination clinical trial design and regulatory synergy claims.

Citation Format: Jianing Fu, Ting-Chao Chou. Simple, efficient, and quantitative approach for determination of synergism, additive effect, and antagonism of drugs in vivo using combination index method: a proposition for clinical protocol design and regulatory synergy claims [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4554A. doi:10.1158/1538-7445.AM2017-4554A
Abstract 4568: A novel TGF-beta receptor I kinase inhibitor demonstrated to be an effective immuno-therapy for cancer
Authors: Xu Zusheng , Lou Yangtong , Chen Li , Zeng Kun , Sun Qingrui , Qu Yingying , Wang Wei , Wu Xiaodong , Ge Xiaomei , Gu Ying , Zhang Yingjia , Zhou He
Published: July 2017

TGF-beta (TGF-β) pathways, despite serving as tumor suppressor in early stage of carcinogenesis, have shown be one of the key tumor-promoting factors in later-stage tumor progression. TGF-β receptor I kinase (TGF-βR1) inhibitors have shown great potentials in their anti-tumor and anti-metastasis efficacy in preclinical studies and some of them are being tested in the clinical settings. A specific TGF-βR1 inhibitor YL-13027 was developed at Yingli Pharmaceutical. YL-13027 possesses good ADME and PK properties and shows potent inhibition of the TGF-β and receptor interaction both in cell-free and cell-based assays. Functionally, in vitro Treg differentiation assay demonstrated the significant inhibition of the induction of FoxP3 expressing cells as well as inhibiting the secretion of IL-10 by YL-13027. In murine colon cancer CT26 model, YL-13027 was well tolerated and significantly inhibited tumor progression with dose dependency. More mechanistic studies are on-going. These data suggest that YL-13027, a novel TGF-βR1 inhibitor, represents a promising and safe immune modulator and shows great potential as a cancer immuno-therapeutics.

Citation Format: Zusheng Xu, Yangtong Lou, Li Chen, Kun Zeng, Qingrui Sun, Yingying Qu, Wei Wang, Xiaodong Wu, Xiaomei Ge, Ying Gu, Yingjia Zhang, He Zhou. A novel TGF-beta receptor I kinase inhibitor demonstrated to be an effective immuno-therapy for cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4568. doi:10.1158/1538-7445.AM2017-4568
Abstract 4576: Targeted human cytolytic fusion proteins: an update
Authors: Barth Stefan
Published: July 2017

Targeted immunotherapeutics, such as antibody drug conjugates (ADCs) or immunotoxins (ITs) represent promising agents for treatment of cancer. Despite their encouraging performance in clinical trials, both ADCs and ITs suffer from disadvantages like stoichiometrically undefined chemical linkage of the cytotoxic payload and potential immunogenicity of bacteria- or plant-derived toxins in humans, respectively. Therefore, in contrast to depleting B cell epitopes from bacterial toxins, we designed a number of targeted recombinant cytolytic fusion proteins, replacing bacterial toxins like Pseudomonas exotoxin A by human enzymes like proteases, kinases, RNases or microtubuli-associated proteins. Binding and biological activities were shown in vitro, ex vivo and in different in vivo models. This presentation will summarize the latest results on the hCFPs generated and demonstrate the efficacy of treatment of CD64-, CSPG4-, and EpCAM-targeting constructs in direct comparison to the corresponding Pseudomonas exotoxin-based immunotoxins. The presentation will conclude with an outlook on future perspective and applications of the next generation of targeted fully human recombinant cytolytic fusion proteins.

Citation Format: Stefan Barth. Targeted human cytolytic fusion proteins: an update [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4576. doi:10.1158/1538-7445.AM2017-4576
Abstract 4591: Specific elimination of invasive and multidrug-resistant cancer cells by an antibody-drug conjugate targeting AXL
Authors: Boshuizen Julia , Koopman Louise A. , Breij Esther C. , Satijn David , Peeper Daniel , Parren Paul W.
Published: July 2017

Upon therapeutic pressure, cancers commonly select for drug-resistant, invasive subpopulations with elevated expression of the receptor tyrosine kinase AXL. Besides the correlation between high AXL expression and induction of epithelial-to-mesenchymal transition, a process known to support metastasis, ample evidence also links AXL to resistance against a variety of targeted therapies, including inhibitors of the MAPK pathway in malignant melanoma and the EGFR pathway in lung cancer.

AXL-107-MMAE (HuMax-AXL-ADC) is a therapeutic antibody-drug conjugate specific for AXL, containing the microtubule disrupting agent monomethyl auristatin E as the cytotoxic payload. AXL-107-MMAE was previously shown to induce potent cytotoxicity in vitro and in vivo, which was dependent expression of AXL on the cell surface.

We evaluated the capacity of AXL-107-MMAE to target AXL-positive MAPK pathway inhibitor resistant tumor cells, using malignant melanoma as a clinically relevant example. First, AXL-107-MMAE was shown to induce cytotoxicity in BRAF-mutant tumor cell lines that showed AXL expression upon acquired resistance to BRAF-inhibitors. In contrast, no cytotoxicity was observed in the BRAF-inhibitor-sensitive, AXL-negative parental cell lines. In heterogeneous tumor cell cultures, treatment with a BRAF-inhibitor selected for AXL-high, MAPK pathway inhibitor-insensitive melanoma cells, which was prevented by combined BRAF-inhibitor and AXL-107-MMAE treatment. Interestingly, we observed marked AXL upregulation in biopsies obtained from patients after they developed resistance to MAPK pathway inhibitors compared to paired pre-treatment biopsies.

The in vivo potential of AXL-107-MMAE in malignant melanoma was demonstrated using BRAFV600E-mutated xenograft model derived from a patient who developed resistance to the BRAF inhibitor vemurafenib in the clinic. This indicates that AXL expression levels in treatment-resistant malignant melanoma are sufficient to induce tumor regression with AXL-107-MMAE, at least in this model system.

These findings merit clinical validation of the targeting of both treatment-naïve and drug-resistant cancers with AXL-107-MMAE, either alone or in combination with other targeted therapies.

Citation Format: Julia Boshuizen, Louise A. Koopman, Esther C. Breij, David Satijn, Daniel Peeper, Paul W. Parren. Specific elimination of invasive and multidrug-resistant cancer cells by an antibody-drug conjugate targeting AXL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4591. doi:10.1158/1538-7445.AM2017-4591