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The lack of reproducibility of preclinical research is a significant and growing problem which slows basic research and leads to fruitless clinical trials. An increasing number of reports have found discrepancies in published preclinical studies across scientific disciplines. For example: * Amgen found that 47 of 53 “landmark” oncology publications could not be reproduced. * Bayer found that their internal results contradicted academic publications in 43 of 67 oncology and cardiovascular projects. * Dr. John Ioannidis and his colleagues found that of 432 publications purporting sex differences in hypertension, multiple sclerosis, or lung cancer, only one data set was reproducible. These studies, and the many others that report similar results, highlight a significant problem in the development of new therapies to treat disease. With increasing reports of discrepancies in preclinical publications, pharmaceutical companies are being forced to re-evaluate their reliance on academic research. In fact, Bayer recently decided to halt nearly two-thirds of target-validation projects.In this session, we'll report the work we have done to address this issue. We will bring together researchers and representatives from funders and publishers to discuss the issue of reproducibility. We will share funder and publisher initiatives to promote reproducibility and also discuss research practices that lead to more reproducible research such as using proper statistical tests, reporting all experimental data, experimental blinding, and identification and validation of research reagents. We'll also share preliminary results from an Arnold Foundation-funded study to replicate the 50 most high impact cancer biology studies from 2010-1012. Reproducibility ResearchAmgen47 of 53 “landmark” oncology publications could not be reproducedBayerinternal results contradicted academic publications in 43 of 67 oncology and cardiovascular projectsDr. John Ioannidis and colleagues432 publications purporting sex differences in hypertension, multiple sclerosis, or lung cancer, only one data set was reproducible Note: This abstract was not presented at the meeting. Citation Format: Gunn William, Elizabeth Iorns, Elizabeth Silva, Brian Nosek. Preliminary results from the Reproducibility Project: Cancer Biology - a replication of 50 high-impact cancer cell biology papers from 2010-2012. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5053. doi:10.1158/1538-7445.AM2014-5053

Background: Natural killer (NK) cell-based adoptive immunotherapy is a promising approach for the treatment of cancer. But, it is difficult to generate the sufficient scale and purity of NK cells, and reliable methods to produce large number of functional NK cells have not been established yet. We have developed novel clinical-grade NK cells expansion method to produce the high purity, large scale and functional NK cells using a combination of recombinant human fibronectin fragment (RetroNectin®) -induced T-cells (RN-T cells), OK-432 and IL-2. We subsequently conducted a Phase 1 clinical study to evaluate the safety and efficacy of this NK cell therapy. In this paper, we address the characteristics of the NK cells elicited by this method and the results of immune monitoring in the clinical trial. Methods: To confirm the significance of RN-T cells as stimulator, we compared the stimulation effects on NK cells between RN-T cells and aCD3-T cells, which stimulated by anti-CD3 mAb only. Next, we analyzed expanded NK cells from PBMCs obtained from 31 cancer patients to verify the efficacy of this method. In the Phase 1 trial, patients with unresectable digestive cancer were enrolled. They received weekly intravenous administration of autologous NK cells elicited by the novel method three times to assess the safety of the number of adoptive cells at 0.5 × 109(cohort1), 1 × 109(cohort 2) and 2 × 109 cells (cohort 3) per dose. The phenotype and cytotoxicity of expanded NK cells were analyzed. For immune monitoring, cytotoxicity of PBMCs and whole blood cytokine levels were examined following NK cell infusion. Results: The stimulation by RN-T cells could induce preferable NK cell proliferation rather than the one by aCD3-T. As results of 31 cancer patients, 688±76-fold expansion was achieved in this system with PBMCs, and the NK cell populations were highly purified (84.7±3.6%) and highly expressed functional markers such as NKG2D (97.3±0.6%) and CD16 (96.8±0.7%). In the Phase 1 clinical trial, no NK cell infusion related severe or unexpected toxicities were observed. The response rate and the disease control rate in 10 per protocol patients were 0% and 50.0%, respectively. Although no clinical responses were observed, the cytotoxicity of PBMCs against K-562 targets increased in most patients (80%). The average of cytotoxic activity of PBMCs increased more than two times after the NK cell infusion. Conclusion: Although no patients receiving the NK cell therapy alone experienced a clinical response, adoptive immunotherapy of the NK cells elicited by the novel method is expected to exert considerable ADCC activity in vivo because of their high expression of CD16. Now, we are conducting clinical trial in which we explore the combination of this novel NK therapy with IgG1 antibodies such as trastuzumab and cetuximab. We also address the ongoing clinical trial in this paper. Citation Format: Takeshi Ishikawa, Naoyuki Sakamoto, Tetsuya Okayama, Kaname Oka, Satoshi Kokura, Mitsuko Ideno, Akiko Kato, Tatsuji Enoki, Masanari Kitagawa, Junichi Mineno, Tomoyo Yasuda, Toshifumi Doi, Yuji Naito, Yoshito Itoh, Toshikazu Yoshikawa. A novel expansion method for functional natural killer cells and its clinical application. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3137. doi:10.1158/1538-7445.AM2015-3137

In previous paper we reported on the much higher selectivity toward cancer cells and tissues of tetra-branched neurotensin peptides (NT4) compared to monomeric NT peptide. We also demonstrated that NT4 can be coupled to many different functional units for cancer cell tracing and drug delivery and can induce tumor growth reduction in animal studies. We then proposed NT4 as promising cancer selective theranostics for different human cancers, including CRC, pancreas adenocarcinoma and urinary bladder cancer. Nonetheless, multimeric binding of tetrabranched peptides, together with the chemical modification produced by coupling to the branched core, might have modified receptor selectivity of NT4 with respect to native monomeric NT and actually we had no conclusive indication on which receptor our branched NT4 peptides were binding to. Data reported in the present paper demonstrate that synthesis of neurotensin sequence in a tetra-branched form induce a switching of receptor selectivity, by decreasing affinity to the NT high affinity receptor NTR1 and contemporarily acquiring binding to additional receptors, which produces a much higher cancer cell selectivity of NT4 with respect to monomeric NT peptides. We demonstrate here that NT4 binds sortilin and SorLa and also acquire the ability to bind different receptors belonging to the Low Density Lipoprotein Receptor Related Protein (LRP) family as well as heparin and other Heparan Sulfate Proteoglycans (HSPG). The much higher binding of NT4 in respect to native NT to either cancel cell lines or human cancer surgical samples, as well as the higher selectivity toward human cancer tissues of NT4 is due to binding to different membrane receptors, which are very selectively expressed by many different human cancers. Moreover, our results confirm that sulfated proteoglycan can mimic the ligand binding site of different LRP receptors and indicate that targeting of multiple LRP receptors together with sulfated proteoglycans produce an extremely high selectivity towards many different human cancers. Citation Format: Luisa Bracci, Chiara Falciani, Jlenia Brunetti, Barbara Lelli, Niccolò Ravenni, Luisa Lozzi, Lorenzo Depau, Alessandro Pini. Targeting different LRP receptors and sulfated proteoglycan by branched neurotensin provide high cancer selectivity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5625. doi:10.1158/1538-7445.AM2013-5625

Background: To facilitate drug discovery and development in triple-negative (TNBC) and basal-like (BLBC) breast cancers, an integrated knowledge space was developed through standardizing and linking information reported in scientific publications. The tool was designed to 1) track and consolidate knowledge around TNBC and BLBC; 2) centralize computational resources on molecular profiles, disease models, drug effects and clinical outcomes; 3) establish a computational platform to facilitate identification of drug targets and biomarkers; 4) enable hypothesis generation for novel uses of pre-existing compounds including new indications and combinations. Material and Methods: Querying PubMed with “(basal-like or triple-negative) breast cancer” generated 1200 hits. First, text-mining techniques were employed to filter publications for curation. Second, information extracted from biomedical literature and public domain databases was standardized with control vocabularies. Third, interactions originally presented in natural language were translated into a well-structured format according to specific semantic rules suited for computational purposes. The concepts and associated interactions were captured in a database and could be browsed as lists, tables and interactive networks. Results: Based on the initial assessment of the 1200 abstracts, 500 papers were selected for further analysis. About 1400 unique concepts were extracted and categorized: 1) 600 molecular components such as DNA loci, SNP, mRNA, microRNA, protein and complexes; 2) 120 cellular processes; 3) 100 model systems such as cell lines, xenograft, and tissues; 4) 220 compounds and regimens; 5) 180 clinical and non-clinical parameters such as survival and chemoresistance; 6) 264 disease definitions. The most implicated genes in TNBC and BLBC were EGFR, BRCA1, TP53, CK5/6, AR, TP63, KRT14, CDH1, AKT1, CCND1, VEGF, PTEN, CD44, NFKB1 etc. There were 80 genes reported related to TNBC and BLBC by more than 3 papers. Using the published gene expression datasets of breast cancers, the 80-gene set proved to be a highly accurate classifier for BLBC and non-BLBC. The most researched cellular processes included epithelial to mesenchymal transition (EMT), cell migration/metastasis, apoptosis, cell proliferation, angiogenesis, beta-catenin/WNT, PI3k/AKT. The most studied experimental therapies included PARP inhibitors, cisplatin, dasatinib, doxorubicin and EGFR inhibitors. Concepts and interactions were visualized in networks. For example, the EMT network comprised 110 nodes and 162 interactions based on 19 references, and the TNBC network contained 514 nodes and 874 interactions from 217 papers. Discussion: The number of research papers on TNBC and BLBC are growing rapidly with 30–50% increase annually. TNBL provides a platform for the research community to standardize scientific findings and exchange knowledge efficiently. Through its powerful semantic technology and robust visualization capability, it offers integrated and comprehensive views of TNBC and BLBC which are not possible in the scope of any single research paper. In addition, it facilitates high quality hypothesis generation through consolidating up-to-date information from all possible sources in a computable format. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-04-02.

Continuous cell lines are widely used, but can result in invalid, irreproducible research data. Cell line misidentification is a common problem that can be detected by authentication testing; however, misidentified cell lines continue to be used in publications. Here we explore the impact of one misidentified cell line, KB (HeLa), on the scientific literature. We identified 574 articles between 2000 and 2014 that provided an incorrect attribution for KB, in accordance with its false identity as oral epidermoid carcinoma, but only 57 articles that provided a correct attribution for KB, as HeLa or cervical adenocarcinoma. Statistical analysis of 57 correct and 171 incorrect articles showed that the number of citations to these articles increased over time. Content analysis of 200 citing articles showed there was a tendency to describe the cell line in accordance with the description in the cited paper. Analysis of journal impact factor showed no significant difference between correct and incorrect groups. Articles using KB or citing that usage were most frequently published in the subject areas of pharmacology, pharmacy, oncology, and medicinal chemistry. These findings are important for science policy and support the need for journals to require authentication testing as a condition of publication. Cancer Res; 77(11); 2784–8. ©2017 AACR.

Purpose: We plan to use a tissue microarray-based microRNA(miRNA) detection by in situ hybridization with LNA probe and support vector machine on base of data mining to construct the Prognostic model for breast cancer and triple negative breast cancer (TNBC). Methods: Combining the BreastMark miRNA database and papers published, we chose candidate miRNAs. Then a tissue microarray-based miRNA detection in situ hybridization with LNA probe was used to detect miRNA expression in 445 breast cancer tissue. Univariate analysis with Kaplan-Meier identified independent prognostic factors, 445 patients was divided into the training set and validation set (70% VS 30%) by software R(version 3.2.3), then support vector machine on base of data mining was used to construct the Prognostic model for breast cancer and triple negative breast cancer via software R and predict.svm(e1071). Results: 15 miRNAs were detected from 445 breast cancer tissue and univariate analysis with Kaplan-Meier identified significant prognostic miRNAs: miR-361-5p, miR-301a, miR-223, miR-421, miR-454, miR-493 were the independent prognostic factors for DFS. By using data mining technique, we combined clinical factors(stage, tumor size, lymphnode status, molecular subtype) and miRNAs mentioned above to establish the personalized predictive mathematical models SVM-BC and SVM-TNBC. In the training set and validation set of SVM-BC model, the AUC value of ROC curve for DFS were 0.86,0.83. The total accuracy,sensitivity,specificity of SVM-BC model for DFS were 94%,73%,99% and 84%,57%,92% in the training set and validation set. The SVM-BC model can divide breast cancer patients into high-risk and low-risk groups, and 5-year survival rates for low-risk patients were higher than that for high-risk patients(96.8% VS 39.3%, p In the training set and validation set of SVM-TNBC model, the AUC value of ROC curve for DFS were 0.84,0.86. The total accuracy,sensitivity,specificity of SVM-TNBC model for DFS were 90%,71%,97% and 84%,55%,94% in the training set and validation set. The SVM-TNBC model can divide breast cancer patients into high-risk and low-risk groups, and 5-year survival rates for low-risk patients were higher than that for high-risk patients(93.9 % VS 23.5%, p Conclusion: The Prognostic model basing of tissue microarray-based miRNA detection in situ hybridization with LNA probe and support vector machine for prognosis can effectively predict the prognosis of breast cancer and TNBC. Citation Format: Cao Z, Yao L, Hu X, Zhang Z, Shao Z. In-situ hybridization of microRNA and support vector machines–based prognostic classifiers for breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-05-37.

Emerging sets of single-cell sequencing data makes it appealing to apply existing tumor phylogeny reconstruction methods to analyze associated intratumor heterogeneity. Unfortunately, tumor phylogeny inference is an NP-hard problem and existing principled methods typically fail to scale up to handle thousands of cells and mutations observed in emerging single-cell data sets. Even though there are greedy heuristics to build hierarchical clustering of cells and mutations, they suffer from well-documented issues in accuracy. Additionally even when “optimal” solutions are feasible, existing approaches only provide a single “most likely” tree to depict the evolutionary processes that may result in an observed collection of cells and mutations. To make matters worse, the vast majority of single-cell sequencing data sets are transcriptomic and as a result, suffer from considerable variation in coverage across mutational loci. In this paper, we introduce Trisicell, a computational toolkit for scalable tumor phylogeny reconstruction and validation from single-cell genomic, exomic or transcriptomic sequencing data. Trisicell has three components: (i) Trisicell-DnC, a new tumor phylogeny reconstruction method from genotype matrices derived from single-cell data, (ii) Trisicell-ConT a new algorithm for constructing the consensus for two or more tumor phylogenies - which may be built through the use of different data types on the same set of cells, or built through the use of different methods on the same data, and (iii) Trisicell-PF, a new partition function method for assessing the likelihood of any user-defined subtree/set of cells to be seeded by a given set of mutations in the phylogeny. Collectively, these tools provide means of identifying and validating robust portions of a tumor phylogeny, offering the ability to focus on the most important (sub)clones and the genomic alterations that seed the associated clonal expansion. We applied Trisicell to a panel of clonal sublines derived from single-cells of a parental mouse melanoma model on which we performed both whole exome and whole transcriptome sequencing. The tumor phylogenies of the clonal sublines built on exomic and transcriptomic mutations by Trisicell-DnC, were shown by Trisicell-ConT to be highly similar and the subtrees comprised of phenotypically similar clonal sublines were shown to be strongly associated by Trisicell-PF to their seeding mutations. In addition, we applied Trisicell to single-cell whole transcriptome sequencing data from a tumor derived from the same parental melanoma cell line, which was subjected to anti-CTLA-4 immunotherapy. The phylogenies generated from both studies featured distinct subtrees, strongly associated with phenotypes including cell differentiation status, tumor growth and therapeutic response. These results suggest that Trisicell can be used for scalable tumor phylogeny reconstruction and validation through both single-cell and clonal-subline sequencing data, which may reveal strong phenotypic associations. In particular, they suggest that the developmental status and phenotypic intratumoral heterogeneity of melanoma originates from observable subclonal variation. Citation Format: Farid Rashidi Mehrabadi, Salem Malikić, Kerrie L. Marie, Eva Pérez-Guijarro, Erfan Sadeqi Azer, Howard H. Yang, Can Kızılkale, Charli Gruen, Huaitian Liu, Christina Marcelus, Aydın Buluç, Funda Ergün, Maxwell P. Lee, Glenn Merlino, Chi-Ping Day, S. Cenk Sahinalp. Trisicell: Scalable Tumor Phylogeny Reconstruction and Validation Reveals Developmental Origin and Therapeutic Impact of Intratumoral Heterogeneity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB019.

The human leukocyte antigen class I (HLA-I) genes shape our immune response against pathogens and cancer. Certain HLA-I variants can bind a much wider range of peptides than others, a feature that could be favorable against a range of viral diseases. However, the implications of this phenomenon on cancer immune response is unknown. In this paper, we quantified peptide repertoire breadth (or promiscuity) of a representative set of HLA-I alleles, and found that cancer patients that carry HLA-I alleles with high peptide binding promiscuity are characterized by significantly worse prognosis after immune checkpoint inhibitor treatment. This trend can be explained by a reduced capacity of promiscuous HLA-I molecules to discriminate between human self and tumor peptides, yielding a shift in regulation of T-cells in the tumor microenvironment from activation to tolerance. In summary, HLA-I peptide binding specificity shapes neopeptide immunogenicity and the self-immunopeptidome repertoire in an antagonistic manner. It could also underlie a negative trade-off between antitumour immunity and the genetic susceptibility to viral infections. Citation Format: Benjamin Tamas Papp, Máté Manczinger, Gergő Balogh, Balázs Koncz, Leó Asztalos, Lajos Kemény, Balázs Papp, Csaba Pál. Negative trade-off between neoantigen repertoire breadth and the specificity of HLA-I molecules shapes antitumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1907.

Cancer is a major death-causing disease worldwide, and metastasis, a hallmark of cancer, is associated with up to 90% of all cancer-related deaths. Metastasis requires ATP, and the tumor microenvironment (TME) has much higher concentrations of intratumoral and extracellular ATP (eATP) than healthy tissue, implying that eATP may have potential roles in tumor development and progression. Our previous ATP studies, cited by a Nature Review Cancer paper in 20181 showed that eATP is internalized by cancer cell through macropinocytosis, and it promotes cancer cell growth, survival, and drug resistance. However, how intratumoral eATP plays roles in metastasis remains largely unclear. Our 2019 published results show that eATP promotes cancer migration and invasion, initiates epithelial to mesenchymal transition (EMT), alters expression level of EMT related protein markers such as ZEB1, SNAIL, SLUG in human non-small lung cancer cell (NSCLC) lines2. It has been reported that the EMT and cancer stem cells (CSCs) share some molecular links, but the detailed mechanisms involved are still unknown. Recently, we have found the eATP significantly induces the protein expression level of CSC related makers in A549 cells. We have also found that, after A549 cells were treated with 0.5mM and 0.75mM ATP for 6 and 24 hours, the ATP treated A549 cells were able to show significantly increased colony numbers by soft agar colony formation assay. Based on all these results, we hypothesize that extracellular ATP functions as a messenger, a signal facilitator, an energy molecule, and a transcription cofactor to induce CSC-like features to promote metastasis in NSCLC. RNAseq and metabolomics studies are being used to determine the transcriptional and metabolic changes associated with the ATP induced CSC process in NSCLC. The completion of the study will not only unravel eATP's roles in CSC induction and early steps of tumor metastasis, but also potentially provide novel promising therapeutic targets for inhibiting tumor metastasis.(1) Di Virgilio, F., Sarti, A. C., Falzoni, S., De Marchi, E., & Adinolfi, E. (2018). Extracellular ATP and P2 purinergic signaling in the tumor microenvironment. Nature Reviews Cancer, 18(10), 601-618. (2) Cao, Y., Wang, X., Li, Y., Evers, M., Zhang, H., & Chen, X. (2019). Extracellular and macropinocytosis internalized ATP work together to induce epithelial-mesenchymal transition and other early metastatic activities in lung cancer. Cancer Cell International, 19(1). Citation Format: Jingwen Song, Xiaozhuo Chen. Roles of extracellular ATP in inducing cancer stem cell (CSC)-like changes in non-small lung cancer cell line [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3106.

Cancer is a major death-causing disease worldwide, and metastasis, a hallmark of cancer, is associated with up to 90% of all cancer-related deaths. Metastasis requires ATP, and the tumor microenvironment (TME) has much higher concentrations of intratumoral and extracellular ATP (eATP) than healthy tissue, implying that eATP may have potential roles in tumor development and progression. Our previous ATP studies, cited by a Nature Review Cancer paper in 20181 showed that eATP is internalized by cancer cell through macropinocytosis, and it promotes cancer cell growth, survival, and drug resistance. However, how intratumoral eATP plays roles in metastasis remains largely unclear. Our 2019 published results show that eATP promotes cancer migration and invasion, initiates epithelial to mesenchymal transition (EMT), alters expression level of EMT related protein markers such as ZEB1, SNAIL, SLUG in human non-small lung cancer cell (NSCLC) lines2. It has been reported that the EMT and cancer stem cells (CSCs) share some molecular links, but the detailed mechanisms involved are still unknown. Recently, we have found the eATP significantly induces the gene expression level of CSC transcription factors (TFs): SOX2, NANOG and OCT4 in A549 cells in a time-dependent manner. We have also found that, after A549 cells were pretreated with 0.5mM for 6 hrs, the ATP-pretreated A549 cells were able to show significantly increased migration and invasion even in the absence of ATP addition. Based on all these results, we hypothesize that extracellular ATP functions as a messenger, a signal facilitator, an energy molecule, and a transcription cofactor to induce EMT and CSC-like features to promote metastasis in NSCLC. RNAseq and metabolomics studies are being used to determine the transcriptional and metabolic changes associated with the ATP induced EMT and CSC process in NSCLC. The completion of the project will not only unravel eATP's roles in EMT and CSC induction and early steps of tumor metastasis, but also potentially provide novel promising therapeutic targets for inhibiting tumor metastasis. Citation Format: Jingwen Song, Maria Evers, Xiaozhuo Chen. Roles of extracellular ATP in inducing EMT and CSC-like changes to promote early steps of metastasis in non-small lung cancer cell lines [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1542.

Introduction: This paper discusses how cancer arises from chronic inflammation, based on complexity theory. Methods: We reviewed the medical literature to identify cancer types strongly associated with chronic inflammation. We then classified the chronic inflammatory etiologies, determined general mechanisms through which they promote cancer and speculated on network changes involved in transforming cells from physiologic to cancer attractor states. Results: Bacterial and viral infection are a common etiology of chronic inflammation associated cancer and cause 15% of cancer cases worldwide, predominantly Helicobacter pylori, human papillomavirus and hepatitis B and C virus. Other etiologies include parasitic infestations by Opisthorchis viverrini, Clonorchis sinensis and Schistosoma haematobium; autoimmunity in Hashimoto thyroiditis, Sjögren syndrome and celiac disease; local trauma due to gastroesophageal reflux and hot beverages; excess weight; diabetes; Western diet (high fat, low fiber, low consumption of fruit and vegetables); aging and immune system dysfunction. General mechanisms include immune system activation that damages DNA by producing reactive oxygen and nitrogen species and nitrosamines; tumor immune evasion via immune suppression and immune senescence; antigen driven lymphoproliferation; continuous mitotic activity due to repair; synergy with other chronic stressors; creation of a tumor nurturing microenvironment; development of a “runaway” immune system; and microbiome changes that produce carcinogens or activate inflammation. Immune system dysfunction and germ line variations of inflammatory mediators can promote each step. From a network perspective, the usual physiologic state for many cellular processes consists of a delicate balance between stimulating and dampening forces, maintained by inherent network features and evolved control systems. Chronic inflammation may disturb this balance, leading to propagation of network instability throughout the cell, across adjacent tissues and ultimately systemically. This may create identifiable network hierarchies and intermediate states (hyperplasia, metaplasia or dysplasia), but some changes in network and molecular patterns may not alter histology. Ultimately, cells may move to a cancer attractor state. Summary: Chronic inflammation causes cancer by initiating local changes to cellular networks and their microenvironment which facilitate their escape from physiologic states towards intermediate and cancer attractor states. This suggests that early detection and reduction of these inflammatory changes may reduce cancer mortality. Novel treatment options include more diverse treatment combinations, destabilizing existing cancer attractors and their microenvironment, stimulating physiologic pathways that stabilize networks, reducing other chronic stressors and optimizing rational medical care. Citation Format: Nat L. Pernick. How cancer arises from chronic inflammation, based on complexity theory [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5494.

Cancer-associated fibroblasts (CAF), as one of the major components of tumor microenvironment, have a significant role in cancer development and progression. CAF also supports the survival and chemo-induced resistance of cancer cells. Among various signaling pathways involved in cancer cell and CAF interaction, the signal transducers and activators of transcription (STAT3) pathway has been investigated a lot, but its own role in CAF itself is not yet clearly defined. In this paper, we established CAF using MRC5, which is a stable lung fibroblast cell line, by culturing with conditioned medium from lung cancer cells. Then, by using a STAT3 inhibitor, STX-0119, and STAT3 siRNA, we examined whether the blocking of STAT3 pathway in MRC5 affects chemotherapeutic resistance or invasiveness of A549 lung cancer cells. We found out that STAT3 inhibition in fibroblasts did not prevent CAF-induced resistance of A549 cells on proliferation, but it did attenuate the invasiveness and migration of A549 cells. These results suggest that the combination of a low dose of a STAT3 inhibitor, which would be high enough to inactivate the CAF around the tumor cells, and an anticancer drug can be taken as an approach to cancer treatment. Citation Format: Ju-Hee Kang, Ha Jung Lee, Hyun Jin Jung, Min Woo Kim, Seung Hyun Oh. STAT3 pathway in fibroblasts aggravates invasiveness of lung cancer cells in the tumor microenvironment [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr B48.

Increasing evidence shows that tumor clonal architectures are often the consequence of a complex branching process, yet little is known about the expected dynamics and extent to which these divergent subclonal expansions occur. Here, we develop and implement more than 88,000 instances of a stochastic evolutionary model simulating genetic drift and neoplastic progression. Under different combinations of population genetic parameter values, including those estimated for colorectal cancer and glioblastoma multiforme, the distribution of sizes of subclones carrying driver mutations had a heavy right tail at the time of tumor detection, with only 1 to 4 dominant clones present at ≥10% frequency. In contrast, the vast majority of subclones were present at Significance: The model presented in this paper addresses tumor heterogeneity by framing expectations for the number of resistant subclones in a tumor, with implications for future studies of the evolution of therapeutic resistance. Cancer Res; 78(3); 830–9. ©2017 AACR.

The application of Next Generation Sequencing (NGS) technologies in cancer research has accelerated the discovery of somatic mutations; however, it has not mirrored the same progress in the identification of cancer associated germline variation. With the goal to inform future studies on commonly used methods and their degree of success and limitations in identifying germline variants associated with cancer, we performed a review of the English literature for genome-wide germline sequencing studies aimed at identifying new cancer susceptibility genes or variants. A key words search in 2005-April 2017 PubMed yielded 7,349 articles which were checked against the following exclusion criteria: non-cancer (1852), somatic mutations (1764), methods (905), reviews (872), non-human (735), expression (272), targeted sequencing (224), commentaries (170), epigenetic (135), non-English (89), functional (85), clinical studies (51), others (44). The remaining 151 publications were examined in-depth and coded in an Access database according to ~60 fields, capturing information on: publication (journal, year, author, title, abstract), study design (goal; cancer type; population and ethnicity; discovery, technical validation, and replication phases; numbers and characteristics of cases, controls, families used in each phase), sequencing technique (center, technology, coverage, reads aligner, variants caller, QCs), data analysis (variants annotation, candidate genes, filtering strategy, association test), in silico and experimental functional assessment, and key conclusions (main results, challenges, next steps). The discovery phase of the examined papers was largely based on familial cases (77%), exome/genome sequencing of one single family (31%), and restricted to the analysis of candidate genes lists. The distribution of ethnicity for the sequenced cancer patients was 38% European, 11% Asian, 10% African, 5% multi-ethnic, 4% African American, 3% Latino/Hispanic, 3% Middle Eastern, 1% Jewish, and 25% unreported. Breast, prostate, colorectal, and lung cancer represented ~40% of the assessed articles, and the remaining 60% a diverse group of rare cancer types. Most studies (~80%) included a technical validation phase, but without clear success rate. Only 60% of studies included a replication phase in an independent population. Although most studies (~80%) described the identification of multiple germline variants and genes thought to have a role in cancer susceptibility, the supporting evidence was often disparate or not clearly stated. Our findings underscore the importance of establishing standards for reporting filtering strategies and rational for variant identification. We identified untapped potentials in the utilization of more/larger families, the harmonization of results across studies, and the expansion beyond candidate genes. Citation Format: Rolando Barajas, Elise Hoover, Mindy Clyne, Tram Lam, Leah Mechanic, Alisa Goldstein, Elizabeth Gillanders, Melissa Rotunno. A literature review of whole exome and genome sequencing population studies for identifying novel germline mutations in relation to cancer risk [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1600.

Recent advances in sequencing technologies and emerging single cell sequencing promise to profoundly impact cancer studies. One of the most important problems is reconstruction of history of cancer mutations. Existing methods model cells DNA as haplotypes and reconstruct perfect (or almost perfect) phylogeny of mutations from single cell DNA sequencing data. In this paper we propose to reconstruct genotypes (rather than haplotypes) which in absence of repeated and omitted mutations is equivalent to reconstruction of the mutation phylogeny. Single cell genotype reconstruction is proposed to perform in two steps - first the sequencing data are used for imputation of cell genotypes treated as individual human genomes and then the resulted genomes are fitted to perfect phylogeny by eliminating 4-gamete rule violations using integer linear programs. Our simulation results show advantage of single cell genotype imputation over existing methods. Note: This abstract was not presented at the meeting. Citation Format: Alex Zelikovsky, Igor Mandric, Pavel Skums, Ion Mandoiu, Bogdan Pasaniuc, Pramod Srivastava. Imputation of single cell cancer genotypes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-214.

Tumor-associated lymphatics and their connection to the tumor-draining lymph node (TDLN) play important roles in cancer metastasis as well as in modulating the immune microenvironment. They bathe the TDLN in cytokines, exosomes, antigens, and suppressive factors from the tumor microenvironment to alter the anti-tumor immune response. These interactions are difficult to study in vivo, yet currently, these interactions cannot be readily explored in vitro. Here, we developed a perfused paper-based cell culture platform which can recreate the 3-dimensional architecture, stromal component and physiological levels of interstitial flow of the native tissue environment, while keeping the device high-throughput, easy to use, and inexpensive. This platform can sustain the long-term cultures (>3 weeks) of a number of tumor cell lines, which form complex architectures reminiscent of actual tumors, as well as of excised tumor tissue. The platform also supports the function and viability of immune cells (such as T lymphocytes and dendritic cells) and stromal cells (such as lymphatic endothelial cells and fibroblasts). To model the tumor interaction with its TDLN, we created a model in which tumor cells and lymph node stromal cells (LNSC) are cultured in separate but connected compartments under perfusion. To validate this, we have demonstrated preferential tumor migration to preconditioned (vs. unconditioned) LN stroma. The LN stroma compartment also supports co-culture of dendritic cells and T lymphocytes. To validate the modeling of the crosstalk between the tumor and TDLN, we show that tumor conditionioning of the LN primes the LN microenvironment to dampen cytotoxic T cell function. Together, these data highlight the potential of this system for studying complex tumor-host cell interactions in the tumor microenvironment over long periods of time, and paves the way towards an in vitro screening platform for drug screening for drugs that target cells or factors in the tumor microenvironment that serve as barriers to both chemotherapy as well as immunotherapies. Citation Format: RUOLAN ZHOU, Rachel Weathered, Luis Alonzo, Renata Mezyk-Kopec, Melody Swartz. Development and validation of a novel in vitro model of the tumor immune microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1153.

The Pan-Gynecologic (Pan-Gyn) molecular study analyzed data from 2,579 tumors representing five TCGA tumor-type projects: high-grade serous ovarian cystadenocarcinoma (OV), invasive breast carcinoma (BRCA), uterine corpus endometrial carcinoma (UCEC), cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC), and uterine carcinosarcoma (UCS). Principal aims were to identify shared and unique molecular features, clinically meaningful subtypes, and potential future therapeutic targets. We analyzed data of multiple types, including somatic copy number alterations (SCNAs), mutations, DNA methylation, mRNA expression (from mRNA-seq), microRNA (miRNA) expression (from miRNA-seq), long non-coding RNA (lncRNA) expression (from miRNA-seq), and protein expression (from reverse-phase protein arrays; RPPA). We found multiple genomic and epigenomic features that were shared across the Pan-Gyn tumor types and distinguished them from other cancer types. Sixty-one somatic copy number alterations (SCNAs) and 46 significantly mutated genes (SMGs) were found in the Pan-Gyn cohort; 11 of the SCNAs and 11 of the SMGs had not been reported in previous TCGA single-disease papers on the same tumor types. Ten predominant mutation signatures were found that correlated with COSMIC signatures, whereas 10% of the samples had no SMGs. Most of the Pan-Gyn tumor types shared similar miRNA profiles. The exception, ovarian cancer, was extremely different from the rest, and, unexpectedly, the miRNA profiles of basal-like breast cancers closely resembled those of cervical cancers. Protein expression analysis (RPPA) revealed some OV and UCEC samples with the “reactive” signature previously identified in BRCA and shown to be prognostically relevant. It also revealed a subtype with low ER and AR expression levels (important markers for hormone therapy) that spanned all 5 tumor types. Novel lncRNA analysis showed several functionally significant ER-regulated lncRNAs and gene/lncRNA interaction networks. Pathway analysis identified subsets of tumor samples that had high levels of leukocyte infiltration, with potential implications for immunotherapy. Using 16 key molecular features across the Pan-Gyn cancers, we identified five novel cross-tumor and prognostic subtypes with possible clinical implications. Finally, we developed a decision tree that can classify patients into the five subtypes based on just 6 of the 16 molecular features. Those 6 features are potentially assessable in clinical laboratories. Citation Format: Ashton Berger, Anil Korkut, Rupa S. Kanchi, TCGA Pan-Gynecologic Group and Research Network, Gordon B. Mills, Douglas A. Levine, Rehan Akbani. A comprehensive TCGA Pan-Cancer molecular study of gynecologic and breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3303.

Recent studies have demonstrated the involvement of colorectal cancer (CRC) stem cells (CSC) in transformation, cancer progression and metastasis. The main goal of this paper was to examine the molecular mechanisms by which SATB2 induced malignant transformation of colorectal epithelial cells. SATB2 induced malignant transformation and these transformed cells gained the characteristics of CSCs by expressing stem cell markers (CD44, CD133, LGR5 and DCLK1) and transcription factors (c-Myc, Nanog and Sox2). Overexpression of SATB2 in normal colorectal epithelial cells increased cell motility, migration and invasion, which were associated with an increase in N-cadherin and Zeb1, and decrease in E-cadherin expression. SATB2 overexpression also upregulated XIAP and cyclin D1, suggesting its role in cell survival and cell cycle. Furthermore, the expression of SATB2 was positively correlated with β-catenin expression in CRC. In contrary, depletion of SATB2 inhibited cell proliferation, colony formation, cell motility and expression of β-catenin, Snail, Slug, Zeb1 and N-cadherin, and upregulated E-cadherin. Furthermore, SATB2 silencing inhibited the expression of stem cell markers, pluripotency maintaining transcription factors, cell cycle and cell proliferation/survival genes and TCF/LEF targets. Finally, β-catenin/TCF-LEF pathway mediated the biological effects of SATB2 in CSCs. These studies support the role of SATB2/β-catenin/TCF-LEF pathway in transformation and carcinogenesis. Citation Format: Yiming Ma, Wei Yu, Sharmila Shankar, Rakesh K. Srivastava. SATB2/β-catenin/TCF-LEF pathway induces cellular transformation by generating cancer stem cells in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4509.

Routine molecular diagnostic tests have yet to be introduced to guide personalized cervical cancer treatment in contrast to other solid cancers. To assess cancer functional events (CFEs), the RAIDs consortium (Rational Molecular Assessment and Innovative Drug Selection, www.raids-fp7.eu) accrued consecutive tumour tissues, whole blood and sera from 419 cervical cancer patients in 18 centers from 7 European Union countries, between 2013-2016 (BioRAIDs: NCT02428842). Whole exome sequencing (WES) is presently available for the first 98 patients, for 20 CC cell lines (list available in supplementary data) and Reverse Phase Protein Array (RPPA) data for 154 patients with a common core set of 91 patients. By comparing the proportion of mutated tumours for each gene and after correction for multiple testing, we detected no significant difference in the frequency of significantly mutated genes (SMG) between the TCGA and RAIDs datasets. However, alterations in epigenetic regulator genes, which are of potential high clinical interest, are not discussed in the TCGA paper. Mutations, frameshift deletions, insertions or stop codons in MLL2 and MLL3 genes, are present in 17% and 19 % respectively (total: 30%) of the first 98 RAIDs tumours, as well as in all 20 cervical cancer cell lines analyzed. Similar to the TCGA results, frequent mutations in genes coding for acetyltransferases such as EP300/CREBBP (9%) were detected in RAIDs tumours and cell lines (15%/25%). With the exception of SHKBP1, all other novel and previously confirmed genes by TCGA were equally detected in the RAIDs dataset, such as PIK3CA (33%), FBXW7 (14%), NFE2L2 (7%), KRAS (4%), ERBB2 (4%), PTEN (5%), CASP8 (3%). Similarly to TCGA, RAIDs RPPA data defined 3 stable clusters. TCGA and RAIDs cluster 1 regrouped proteins frequently associated with EMT (epithelial mesenchymal transition). Significant features of RAIDs cluster 1 were high phospho-YAP (p= 3.11e-06) and phospho-Met (p=4.74e-03), high Met (p=2.42e-11), high Notch (p=4.14e-17), low E-cadherin (p=2.41e-06) as well as significant levels of the phosphorylated forms of EGFR, HER2-3 and AKT and high PD-L1 and B7-H4 expression. While TCGA cluster 2 had been associated with “Hormone” signaling, RAIDs cluster 2 was associated with “DNA damage” signaling. Antibody selection was not identical between TCGA and RAIDs and further comparisons are warranted. In RAIDs, pre-treatment activation of DNA repair proteins (Rad50, Phospho-Chk2, Phospho-DNA-PK, Phospho-FANCD2, Phospho-53BP1) together with low levels of Ki67 (proliferation) appeared predictive for a complete response to standard radio-chemotherapy. This needs validation on an independent population. RAIDs and TCGA cluster 3 were associated with p38 MAPK and PI3K signaling. The RAIDs consortium's future objectives are to better define actionable CFE in relation to outcome and patient's quality of life, allowing the focus on relevant CFEs for patients at high risk. Citation Format: Maud Kamal, Els Berns, Windy Luscap Rondof, Leanne De koning, Gemma Kenter, Attila Kerezst, Marina Popovic, Choumouss Kamoun, Balazs Balint, Suzy Scholl, RAIDs Consortium. RAIDs: Future prospects for innovative targeted treatments in cervical cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3686.

Background: Colorectal cancer (CRC) is one of the most frequent forms of cancer worldwide. Cells with abnormal growth have the ability to invade or spread to other parts of the body (metastasis). Metastasis is the most common cause of death in CRC patients and activation of epithelial-mesenchymal transition (EMT) triggers metastasis. Thus, it is important to understand the mechanisms of EMT to increase the survival rate of CRC. MicroRNA-17 (miRNA-17) has been proven to be significantly higher in CRC tissues than normal tissues. However, there are few papers about the role of miRNA-17 in CRC metastasis. Therefore, in this study, we evaluated the mechanism underlying miRNA-17-5p related EMT in colon cancer cells. Methods: Expression level of miRNA-17-5p as well as target mRNA were analyzed in HCT-15, HT29, LoVo, SW480 and COLO205 cell lines. In order to evaluate the correlation between miRNA-17-5p and EMT phenomenon, selected cell lines were transiently transfected either miRNA-17-5p mimic or miRNA-17-5p inhibitor and then phenotyping studies were conducted. Moreover, to find out the direct target of miRNA-17-5p, we performed AGO2 immunoprecipitation in each colon cancer cell lines. Results: Real-time PCR revealed that miRNA-17-5p expression reversely correlated with vimentin expression in five colon cancer cell lines. Overexpression of miRNA-17-5p inhibited vimentin expression, cell migration and cell invasion in both LoVo and HT29 cells. On the other hand, inhibition of miRNA-17-5p promoted vimentin expression, cell migration and cell invasion in the same parallel cell lines. Through AGO2 immunoprecipitation analysis, we observed that miRNA-17-5p directly bind to vimentin 3'UTR to influence its stability and expression at the transcriptional level. Conclusion: Our data demonstrated that down-regulated miRNA-17-5p promotes colon cancer cell migration and invasion by regulating vimentin expression. These findings propose that miRNA-17-5p may serve as a potential prognostic biomarker in colorectal cancer. Citation Format: Tea Won Kim, Yeo Song Lee, Hye Kyung Hong, Su Jeong Song, Nak Hyeon Yun, Yong Beom Cho. Downregulation of microRNA-17-5p promotes EMT by targeting vimentin in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 492.

Small molecule inhibitors of cyclin-dependent kinases (CDK) 4/6 have created new opportunities for the treatment of advanced hormone-receptor positive (HR+) breast cancer and show promise in other malignancies. Three CDK4/6 inhibitors, palbociclib (PD0332991; Ibrance), ribociclib (LEE011; Kisqali), and abemaciclib (LY2835219), which have been approved or are in late stage trials, are reported to be broadly similar although recent data suggest that abemaciclib has distinct single-agent activity in patients and a unique adverse effects profile. Differences in pharmacokinetics and relative potency for CDK4 versus CDK6 are postulated to account for these differences. In this paper, we use molecular and functional profiling by mRNA sequencing, mass spectrometry-based proteomics, and GR-based dose-response assays to obtain complementary views of the mechanisms of action of CDK4/6 inhibitors. We show that abemaciclib, but not ribociclib or palbociclib, is a potent inhibitor of kinases other than CDK4/6, including CDK1/Cyclin B, which appears to cause arrest in the G2 phase of the cell cycle, and CDK2/Cyclin E/A, which is implicated in resistance to palbociclib. Whereas ribociclib and palbociclib induce cytostasis, and cells adapt to these drugs within 2-3 days of exposure, abemaciclib induces cell death and durably blocks cell proliferation. Abemaciclib is active even in retinoblastoma protein (pRb)-deficient cells in which CDK4/6 inhibition by palbociclib or ribociclib is completely ineffective. The degree of polypharmacology of small molecule drugs is increasingly viewed as an important consideration in their design, with implications for efficacy, toxicity, and acquired resistance. In the case of CDK4/6 inhibitors, we propose that abemaciclib polypharmacology elicits unique molecular responses that are likely to be therapeutically advantageous. More generally, we propose that multi-omic approaches are required to fully elucidate the spectrum of targets relevant to drug action in tumor cells. We expect such understanding to assist in stratifying patient populations and ordering sequential therapies when resistance arises. Citation Format: Hafner M, Mills CE, Subramanian K, Chen C, Boswell SA, Everley RA, Juric D, Sorger PK. Advantageous polypharmacology of abemaciclib revealed by omics profiling of CDK4/6 inhibitors [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD4-02.

It is now accepted that breast cancer is not a single disease, but instead is a spectrum of tumor subtypes with distinct cellular origins, somatic changes and somewhat predictable clinical behaviors. Prognostic factors such as stage and grade, and predictive factors such as hormone receptors and HER2 are the main features upon which clinical decision-making has traditionally been based. Gene expression data coming from DNA microarrays, and now sequencing data, have provided additional insights into the biology of breast cancer and has resulted in the development of a number of clinically useful assays. Our work on breast tumors using genomic analyses has led to a new molecular taxonomy that identifies at least five subtypes of breast cancers (Luminal A, Luminal B, HER2-enriched, Basal-like and Claudin-low). Known as the “intrinsic subtypes," these groups have revealed critical differences in incidence, survival, metastatic site specificity and response to treatment. Importantly, the information provided by the intrinsic subtypes complements the information provided by classical clinical-pathological markers, and adds value beyond estrogen receptor (ER) status, HER2 status and stage. In addition to the intrinsic subtypes, a number of other prognostic and predictive gene expression-based assays have been developed including the two most widely used clinical assays that are OncotypeDX and Mammaprint. Over the last year, at least six broad-scale papers have appeared that describe the application of massively parallel sequencing to hundreds of breast tumors in order to provide a comprehensive set of somatic mutations, methylation patterns, and DNA copy number changes. When these data are combined together, a highly complex picture of the somatic genetic events driving breast cancer pathogenesis have been revealed. Most importantly, new disease entities have been identified, and the genetic causes of most of the previously known breast cancer disease subtypes identified. Among the many examples of mutations possibly causally associated with a given subtype include somatic mutations highly associated with the Luminal A subtype; specific examples include 25/27 PIK3CA E545K mutations being seen in Luminal A tumors, as well as 13/13 intron 4 CA deletion mutants in GATA3. Alternatively, a number of mutations (TP53 and RB1) and copy number changes are uniquely associated with the Luminal B subtype including GATA3 exon 5 frameshift mutations. Some of these somatic mutations (AKT1, PIK3CA, PIK3R1) and DNA copy number changes (HER2, FGFRs, PTEN, INPP4B and MDM2) may also be predictive of response to targeted therapeutics, with most of these alterations being either retrospectively, and/or prospectively, tested in the clinic. Other genomics findings with therapeutic potential include the discovery that a subtype of breast cancer (i.e. the Basal-like subtype) shows significant genetic and mRNA expression similarities with Serous Ovarian Cancers; these similarities include TP53 mutation (>85% with almost no other commonly mutated genes), wide spread genomic instability, RB1-deficiency or Cyclin E1 amplification, BRCA1 inactivation, cMYC amplification, and high expression of AKT3. These common genetic/genomic features suggest that drug regimens that are used for Ovarian Cancer (i.e. a taxane and cis/carboplatin regimen) may also be effective on Basal-like breast cancers, which is being tested at a number of sites worldwide. Two additional “omic” findings of note include a subset of Luminal B cancers that show a hyper-methylated phenotype and an under representation of PIK3CA mutations, and a strong multi-platform signature of HER2 signaling within the HER2-enriched expression subtype that spans technologies from DNA copy number, to mRNA, to protein, to phopho-protein expression. A clear implication of all these “omic” data is that we are now in the era of personalized medicine, which is posing many new challenges. First and foremost of these challenges is to make biological sense, and then clinical sense, of the very large number of variants seen within any given individual, or any tumor. It is likely that the vast majority of these variants are functionally insignificant, and thus a current computational challenge is the automatic annotation, and functional prioritization, of the variants that are coming from any sequenced cancer genome (be it tumor or normal). A second challenge is what “defines a clinically actionable variant," which typically differs from physician to physician. A third challenge is how does one integrates the findings across multiple “omic” platforms as we know that no platform provides the complete cancer genome portrait. For example, it is still likely that the cell type of origin matters, as the consequences of a BRAF mutation in a melanoma are not the same as this same mutation in a colon cancer. Thus, in the long run, the best diagnostics for personalized medicine will likely be a combination of “omics-based” technologies including gene/protein expression, somatic mutations and DNA copy number changes, which will be synthesized together into objective and predictive computational models. Citation Format: Charles M. Perou. The therapeutic implications of genome sequencing and expression analyses for breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr PL01-03. doi:10.1158/1538-7445.AM2013-PL01-03

Control methods to reduce exposure to the fumonisin carcinogens in maize are critical, especially in rural subsistence populations at an increased risk. The fumonisins are a group of mycotoxins mainly produced by Fusarium spp. They are hepatocarcinogenic to rodents, and have been associated with oesophageal and liver cancer in subsistence maize farming communities around the world. The intact molecule and the free amino group determine the cancer-initiating activity, while the carballylic acid moiety plays an active role in absorption of the fumonisins from the gut. Fumonisin B1 (FB1) is the most abundant naturally occurring fumonisin on maize and maize-based food worldwide and is classified as a Group 2B carcinogen by the International Agency for Research on Cancer (IARC). The Joint FAO/WHO Expert Committee on Food Additives (JECFA) has determined a group provisional maximum tolerable daily intake (PMTDI) for FB1, FB2, and FB3, alone or in combination, of 2 µg/kg body weight/day. In many Sub-Saharan countries, where both maize contamination and maize consumption are high, regulatory mechanisms to control mycotoxin levels, including fumonisin, are either lacking or are not enforced. Therefore, reducing exposure levels by interventions, specifically those based on simple low-cost measures acceptable to these communities, becomes relevant. Although certain physical and chemical methods for control of Fusarium diseases and the fumonsins in maize are currently applied, there are several concerns with regards to the health, safety and environmental risks. Biologically based methods are increasingly being explored, including several approaches pre-harvest (breeding for resistant maize cultivars; introduction of biocontrol microorganisms; application of phenolic plant extracts; expression of antifungal proteins and fumonisin degrading enzymes in transgenic maize cultivars). Postharvest approaches include removal of fumonisins from food by natural clay adsorbents and enzymatic degradation of fumonisins through decarboxylation and deamination by recombinant carboxylesterase and aminotransferase enzymes. This paper will discuss biologically based control methods with special emphasis on the development of food-grade recombinant enzymes and its possible application during the conditioning stage of commercial dry milling of maize. Cultural specific and simple intervention strategies for introduction of these methods to rural communities at risk will also be discussed. Biologically based approaches could be beneficial as control measure to reduce exposure of populations exposed to high levels of this carcinogen. Citation Format: Johanna Francina Alberts, Willem Heber Van Zyl, Wentzel Christoffel Gelderblom. Biological approaches for reducing fumonisin carcinogens in maize [abstract]. In: Proceedings of the AACR International Conference: New Frontiers in Cancer Research; 2017 Jan 18-22; Cape Town, South Africa. Philadelphia (PA): AACR; Cancer Res 2017;77(22 Suppl):Abstract nr B24.

Since Rudolf Virchow’s discovery in 1863 of the link between inflammation and cancer, our knowledge about the physiopathologic processes underlying cancer-related inflammation has steadily increased (1). In their seminal paper in 2000, Hanahan and Weinberg proposed that most—if not all—human cancers have acquired six functional capabilities during their development: self-sufficiency in growth signals, insensitivity to antigrowth signals, tissue invasion and metastasis, limitless replicative potential, sustained angiogenesis, and evading apoptosis (2). This engagingly simple scheme has dictated the cancer research scenario over the past decade. Consequent to the intense experimental work devoted to the cellular and molecular mechanisms underlying carcinogenesis and tumor progression in the twenty-first century, Hanahan and Weinberg added four other capabilities to the scheme: altered metabolism, genetic instability, escape from immunosurveillance, and chronic inflammation (3]) This new point of view emphasized the striking concept that tumors should no longer be viewed as cell-intrinsic genetic diseases. Indeed, it is recognized that cancers arise, progress, and respond to therapy from within a complex microenvironment with which they continuously interact. The tumor microenvironment (TME) is a complex network, which includes soluble factors and components of the extracellular matrix as well as stromal, endothelial, and immune cells. Immune cells and, among them, myeloid cells play important roles in cancer development and can promote or inhibit cancer initiation and progression (4). Among tumor-infiltrating immune cells, macrophages are well-known determinants of cancer-related inflammation and are typically characterized by their remarkable plasticity. This consists of the ability to acquire a wide spectrum of activation states in response to various signals derived from the microenvironment. Classical M1 and alternative M2 macrophages represent the paradigm of this property. Tumor-associated macrophages (TAMs) usually display a so-called “M2-like” phenotype that can foster tumor progression in different ways, namely by promoting genetic instability, angiogenesis, and metastasis and by restraining antitumor adaptive immunity. Notably, TAMs can also play a dual role in the response to conventional antitumor therapies: they can enhance the antineoplastic effect or, in contrast, they can sustain a tumor-promoting response and so foil the anticancer power of these drugs (5). Given their roles in tumor development, a number of macrophage-targeted anticancer approaches are currently being evaluated. They include inhibition of macrophage recruitment and/or survival at tumor sites, functional reprogramming of TAMs to the antitumor M1-like phenotype, and enhancement of killing and/or phagocytosis of cancer cells. Moreover, TAMs express checkpoint proteins that modulate T-cell activation in such a way that they can be targeted by checkpoint-blockade immunotherapies. Also, neutrophils can affect the TME. However, unlike macrophages, they do so by releasing stored and newly synthesized inflammatory mediators. In the “classic,” albeit now obsolete, point of view, neutrophils were viewed as terminally differentiated effector cells that play major roles in the acute inflammatory response and antimicrobial defense. This limited standpoint was challenged by the finding that neutrophils can infiltrate tumors, interact with a number of cell populations, and produce a wide array of cytokines and effector molecules that exert a plethora of effects on tumor behavior (6, 7). Therefore, macrophages and neutrophils are both involved in the regulation of the innate and adaptive immune responses in various inflammatory situations, including cancer. Besides immune cells, innate immunity comprises a humoral arm that includes a variety of molecules, namely complement components, collectins, ficolins, and pentraxins. The role of the humoral arm of the innate immune system in cancer-related inflammation is still being evaluated. PTX3 deficiency was recently found to increase susceptibility to mesenchymal and epithelial carcinogenesis in mice (8). In detail, tumor-infiltrating leukocytes and endothelial cells were found to be a major source of PTX3 and to contribute to PTX3-mediated protection against carcinogenesis. PTX3 deficiency was associated with enhanced macrophage infiltration, proinflammatory cytokine production, angiogenesis, complement C3 deposition, and C5a levels, which suggests exacerbated cancer-related inflammation. Moreover, genetic inactivation of C3 reverted the increased susceptibility to 3-MCA-induced carcinogenesis and macrophage recruitment. PTX3 regulated C3 deposition on sarcoma cells by recruiting the negative complement regulator factor H. In addition, CCL2 inhibition was sufficient to revert the increased susceptibility of PTX3-deficient mice to carcinogenesis and the M2-like phenotype of TAMs. Thus, in 3-MCA sarcomas, unleashed complement activation and increased C5a production associated with PTX3 deficiency is likely to increase the production of CCL2, which in turn recruits tumor-promoting macrophages and favors M2-like polarization. PTX3 deficiency was also associated with increased DNA damage, which is in accordance with the theory that cancer-related inflammation contributes to the genetic instability of tumors. Moreover, the PTX3 gene was found to be highly methylated in some human mesenchymal and epithelial tumors, and these epigenetic modifications were responsible for silencing of PTX3 protein. Thus, an essential component of the humoral arm of innate immunity and regulator of complement activation works as an extrinsic oncosuppressor gene in mice and humans by modulating complement-mediated, macrophage-enhanced, tumor-promoting inflammation [8]. In an increasingly more personalized treatment approach, the more accurate understanding of cancer-related inflammation has led to new therapeutic options that target the TME. These strategies include inhibition of inflammatory mediators or of their downstream signaling molecules, blockage of recruitment/activation of myeloid cells, modulation of their immunosuppressive properties, and re-education of TME. These novel therapeutic strategies could synergize with conventional anticancer treatment and so significantly improve the patient’s clinical outcome and follow-up. Citation Format: Maria R. Galdiero, Martina Molgora, Cecila Garlanda, Gianni Marone, Alberto Mantovani. Tumor-associated myelomonocytic cells as therapeutic targets [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 SY06-01. doi:10.1158/1538-7445.AM2017-SY06-01

Background: Triple-negative breast cancer (TNBC) is a complex heterogeneous disease characterized by the absence of three hallmark receptors: human epidermal growth factor receptor 2 (HER2), estrogen receptor (ER), and progesterone receptor (PR). Compared to other breast cancer subtypes, TNBC is more aggressive, has a higher prevalence in African Americans, and more frequently affects younger patients. Currently, TNBC lacks clinically-accepted targets for tailored therapy, warranting the need for candidate biomarkers. Methods: BiomarkerBase, a biomarker knowledgebase developed by Amplion uses a comprehensive list of synonyms to identify biomarkers registered in the records of clinical trials via the government website clinicaltrials.gov. Under each of the registered breast cancer biomarkers (with the exceptions of HER2, ER, and PR), sub-searches were conducted for clinical trials that explicitly used TNBC (or the full name, triple negative breast cancer) in the title of the biomarkers’ studies. Of note, most trials in this review were completed phase II or III. The selected candidate biomarkers were classified into four FDA biomarker categories (surrogate, prognostic, predictive, or pharmacodynamic (PD)) as well as by location (blood/plasma, cell surface, cytoplasm, or nucleus). Current literature about the biomarkers was further examined using PubMed. There, we sought to find papers that studied how the biomarkers may interact. These markers were further organized into a “cellular protein network” that demonstrates potential connectivity. Results: Blood biomarkers include VEGF/VEGFR and IL-8; cell surface biomarkers include EGFR, IGFBP, c-Kit, c-Met, and PD-L1; cytoplasm biomarkers include PIK3CA/AKT/mTOR-related metabolites (GPC, PCh, Lactate/ glucose), pPKM2, F1,6BP, SPHK1, STAT1/IRF2BP2, S6, p4E-BP1, SUV39H1, PTEN, and ALDH1; and nucleus biomarkers include BRCA1, the glucocorticoid receptor (GR), TP53, and Ki67. Table 1 provides an overview of the examined surrogate, prognostic, predictive, and PD biomarkers in TNBC in addition to current literature found in PubMed that provides support for their potential use. Conclusion: To date, there are no clinically-validated biomarkers for TNBC, which has hindered the development of tailored therapy for both chemosensitive and refractory TNBC. Before validation and use of these biomarkers occurs, stringent-clinical criteria must be met first. The list and cellular protein network provided is an inventory and reference point for promising biomarkers for breakthrough, targeted therapies in TNBC. Citation Format: Brett A. Fleisher, Sihem Ait-Oudhia. A network based analysis for the treatment of triple negative breast 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 5568A. doi:10.1158/1538-7445.AM2017-5568A

Breast cancer is one of the most common malignant diseases in women. Epithelial-mesenchymal transition (EMT) has been documented to play an important role in proliferation, invasion and metastasis of tumor cells as well as drug resistance. Even though the signal transducer and activator of transcription 3 (STAT3) is not a master transcription factor of EMT, STAT3 is involved in the regulation of EMT-related gene expression. However, it remains unclear whether targeted inhibitors of STAT3 affect EMT-mediated proliferation, migration, invasion and drug resistance of tumor cells. In this paper, we investigated the effects of STAT3 and its interaction with Twist, a master transcription factor, in EMT program and subsequent changes in proliferation, migration and invasion of breast cancer cells by interfering STAT3 signaling pathway with different strategies such as STAT3 inactivation and STAT3 silencing. Furthermore, we explored the role of inhibiting STAT3 phosphorylation in the EMT regulation of the sensitivity of tumor cells response to doxorubicin. The results showed that the expression and activation of STAT3 correlated with cell phenotype and invasive ability of breast cancer cells. IL-6 induced phosphorylation of STAT3 can promote the EMT of breast cancer cells. WP1066 completely inhibited STAT3 autophosphorylation and IL-6-induced phosphorylation in breast cancer cells. WP1066 reversed EMT to MET through inhibiting the phosphorylation of STAT3 in breast cancer cells and significantly inhibited the proliferation and invasion in breast cancer cells. Silence STAT3 with siRNA also reversed EMT to MET in breast cancer cells. The expression of EMT transcription factor Twist1 was positively correlated with the activation of STAT3 but not with the expression of STAT3. WP1066 increased doxorubicin antitumor activity through inhibiting the STAT3 phosphorylation. These data provide a proof of concept that targeted inactivation of STAT3 can inhibit proliferation and invasion of breast cancer cells and reduce the resistance of Doxorubicin through reversion of EMT. Support by Natural Science Foundation of China (No.81472489) and Science and Technology Development Plan of Shandong (No.2014GSF118040) Citation Format: Yi Ding, Yu Jiang, Wenqing Li, Shurong Liu, Zhong Lu, Lihong Shi, Xuejian Wang, Jian Zhang, Lihua Wang. Targeted inactivation of STAT3 inhibits proliferation and invasion of breast cancer cells and enhances antitumor activity of doxorubicin through reversion of EMT [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 LB-082. doi:10.1158/1538-7445.AM2017-LB-082

A large number of pre-clinical and clinical studies have strongly implicated that combining cancer-derived neo-antigen vaccines with checkpoint control inhibitors will enhance priming and expansion of tumor-specific naïve and memory T-cells resulting in superior efficacy and durability of response. The neo-antigens, derived from somatic mutations are prime candidates for cancer vaccines, not subjected to host’s central and peripheral tolerance. Identifying potential T-cell engaging neo-epitopes among a large number of somatic mutations is like finding a specific needle in a stack of needles. Currently, the available T-cell neo-epitope prioritization pipelines rely primarily on two attributes - the class-I HLA-binding affinity of the mutant peptide compared to the wild-type counterpart, and the level of expression of the mutated gene in tumor cells. The higher the binding differential, and higher the expression level of the mutant allele, the greater is the likelihood for the peptide to be presented on antigen-presenting cells. These approaches however fall short of predicting whether the HLA-bound peptide will engage T-cells by binding to T-cell receptors (TCRs). We have developed a novel algorithm to predict the binding of HLA-peptide complexes to TCRs by analyzing the physico-chemical composition of the amino acids and their positional biases in the 9-mers from crystal structures of HLA-peptide-TCR complex. We applied machine learning approach to build a classification model that can predict whether a given 9-mer peptide is a TCR-binder or not by identifying whether an amino acid at a given position carries key features that will facilitate interaction with the TCR. We applied this approach to positive and negative TCR interactions selected from Immune Epitope data base (IEDB). We tested multiple classification approaches and found that Random Forest and ClassificationviaRegression methods provided the best performance. We achieved more than 99% accuracy at 10-fold cross validation on both training and unseen test datasets. We further validated our model using positive and negative peptides curated from published papers reporting clinical trial results of checkpoint control inhibitors. The performance of the two classification models was evaluated on two different TCR-binding assays - dextramer binding and IFN-γ release. The ClassificationviaRegression method showed a higher positive predictive value for the dextramer-binding assay, whereas the Random Forest method showed a higher positive predictive value for the IFN-γ ELISPOT assay, suggesting subtle differences between the two classification methods. The inclusion of the TCR binding step to our T-cell neo-epitope prioritization pipeline increased accuracy of prediction, reduced false positives and selected potential neo-epitopes to a manageable number for testing in cell-based assays. Citation Format: Priyanka Shah, Kiran V. Paul, Malini Manoharan, Amitabha Chaudhuri, Ravi Gupta. A novel algorithm to identify TCR-binding somatic mutations from human cancers [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 3579. doi:10.1158/1538-7445.AM2017-3579

Resistance to current chemotherapeutic or radiation-based cancer treatment strategies is a serious concern. Cancer stem cells (CSCs) are typically able to evade treatment and establish a recurrent tumor or metastasis, and it is these that lead to the majority of cancer deaths. Therefore, a major current goal is to develop treatment strategies that eliminate the resistant CSCs as well as the bulk tumor cells in order to achieve complete disease clearance. Aldehyde dehydrogenases (ALDHs) are important for maintenance and differentiation of stem cells as well as normal development. There is expanding evidence that ALDH expression increases in response to therapy and promotes chemoresistance and survival mechanisms in CSCs. This perspective will discuss a paper by Cojoc and colleagues recently published in Cancer Research, that indicates ALDHs play a key role in resistance to radiation therapy and tumor recurrence in prostate cancer. The authors suggest that ALDHs are a potential therapeutic target for treatment prostate cancer patients to limit radiation resistance and disease recurrence. The findings are consistent with work from other cancers showing ALDHs are major contributors of CSC signaling and resistance to anti-cancer treatments. This perspective will address representative work concerning the validity of ALDH and the associated retinoic acid signaling pathway as chemotherapeutic targets for prostate as well as other cancers.

We appreciate the interests of Stauber and colleagues in our study-identifying a small molecule Taspase1 inhibitor (TASPIN) NSC48300 that disrupts cancer cell growth (1). First, regarding the expression level of Taspase1 in 293T cells: Taspase1 was first purified from 293T cells based on the presence of potent proteolytic activity (2). Therefore, the claim that 293T has no measurable expression of Taspase1 is incorrect. The activity of Taspase1 in 293T cells is quite evident as the dual fluorescent proteolytic reporter (GFP 2XNES-MLL(2500-2900)-3XNLS dsRED2) is efficiently processed in 293T cells, resulting in red fluorescence in the nucleus and green fluorescence in the cytosol (ref 1. Fig. 3). This assay was established in 2004 and utilized to screen for bioactive TASPINs (1). Second, regarding the Taspase1 cleavage consensus motif (IXQL(V)D/G) and the importance of hydrophobic residue at the P5 position: To fully understand the biological function of Taspase1, we have generated Taspase1 knockout mice (3) and tried various in silico methodologies, hoping to identify additional Taspase1 substrates. Disappointingly, all candidate substrates identified through in silico approaches fail to pass our established in vitro and in vivo Taspase1 cleavage assays. Hence, we have discarded such an approach for many years. We were quite intrigued by the success using an in silico approach by Bier et al. in identifying many Taspase1 substrates, among which USF2 was best characterized (4). As MLL1, MLL2, TFIIA, and ALF are bona fide Taspase1 substrates identified so far (3, 5), we wished to expand the Taspase1 substrate repertoire and thus examined whether USF2 is a Taspase1 substrate. As pointed out by Stauber and colleagues, USF2 does not carry a hydrophobic residue at P5. We first performed an in vitro cleavage assay using in vitro transcribed and translated full length USF2. Unlike MLL and TFIIA that are efficiently processed by purified recombinant Taspase1 in vitro, USF2 was not cleaved. We then examined the cleavage of USF2 in vivo using two different assays employing Taspase1+/+ and Taspase1-/- mouse embryonic fibroblasts (MEFs). In consistence with our in vitro data, we did not detect processing of endogenous and exogenously expressed USF2 in Taspase1+/+ cells. Third, as NSC48300 is an arsenic acid containing compound, it is foreseeable that it has additional targets other than Taspase1, which was discussed in our paper (1). Accordingly, collaborative efforts are undertaken by the Chemical Biology Consortium through the National Cancer Institute to identify additional TASPINs for potential clinical application.

Recent studies have demonstrated an unexpected connection between aberrant transcription of noncoding RNA (ncRNA) in tumors and innate immune system activation in the tumor microenvironment (TME). Such ncRNA are often of unknown function and may consist of typically silenced interspersed elements, satellite repeats, and endogenous retroviruses (ERVs). For instance, satellite RNA from the pericentromere (particularly HSATII) has been shown to be abundantly transcribed in several solid tumors, including the often lethal pancreatic ductal adenocarcinoma, yet is virtually silent in normal tissue. The genomic DNA repetitive regions this RNA is transcribed from frequently expand during tumerigenesis. Using a novel computational approach, we have shown that, unlike ncRNA expressed under normal conditions, a set of such repetitive elements, abundantly expressed in tumors, display patterns typically associated with viruses. We therefore predicted they are immunogenic, particularly HSATII. In a novel, theory-experiment collaboration between the laboratories of Professors Benjamin Greenbaum and Nina Bhardwaj, the most significant set of these ncRNA have been validated as immunogenic (HSATII and murine GSAT), capable of activating antigen presenting cells - HSATII stimulated production of IL-6, IL-12 and TNFalpha (Tanne, et al., PNAS, 2015). At the same time a set of recent papers has shown that ERV transcription may be a predictor of immunotherapy response in melanoma. Hence, it is critical to profile key “immunogenic” ncRNA (i-ncRNA) in the tumor microenviroment, understand which immune pathways different sets of i-ncRNA activate, and assess the link between the specific pathways activated, prognosis, and immunotherapy. Our preliminary work on melanoma transcriptomes has shown that activation of endogenous elements implies better prognosis. We further profile the landscape of activation for i-ncRNA candidates. We have currently demonstrated a key set of ncRNAs preferentially expressed in cancer cells have sequence features that are immunostimulatory in humans. Quantitatively, our analysis uses a new, unique set of computational methods to study the innate immune system in the TME, originally designed to find motifs in viruses targeted by innate immune receptors (Greenbaum, et al., PNAS, 2014). Our methods for motif characterization utilize a novel mathematical approach we created based on transfer matrix approaches in statistical physics, and are here utilized for the purpose of discovering immunogenic nucleic acids in the tumor microenvironment. They offer several advantages over previous approaches. In particular they are far more computationally efficient, allowing the discovery of anomalous, potentially immunostimulatory patterns of motif usage in far larger sequence datasets than was previously practical. Citation Format: Alexander Solovyov, Antoine Tanne, David Ting, Arnold Levine, Nina Bhardwaj, Benjamin Greenbaum. The landscape of immunostimulatory RNA transcription. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 531.

Tumor-initiating stem-like cells (TICs) are a minor population in bulk tumors that play a critical role in tumor recurrence and therapy-resistance. We previously showed that a key stemness marker Nanog is upregulated that regulates self-renewal and pluripotency of embryonic stem cells and TICs. We demonstrated that alcohol-mediated activation of Toll-like receptor 4 (TLR4) by endotoxin resulted in increased expression of pluripotency stem cell transcription factor NANOG that metabolically reprograms tumor-initiating stem-like cells (TICs: cancer stem cells) via inhibition of oxidative phosphorylation and activation of fatty acid oxidation in our recently accepted Cell Metabolism paper. Discovery of a drug that specifically targets TICs would be a vital goal for cancer therapy. To identify selective TIC inhibitors, we conducted a high throughput screening of an FDA-approved drug library using combination of Nanog promoter-GFP-based screening with viability-based screening and their combination screening of each hit compound. Our high-throughput screening identified the best combination of repurposed FDA-approved drugs: all-trans retinoic acid (ATRA) and the histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) that repressed NANOG and induces apoptosis of TICs. Treatment of ATRA inhibits self-renewal ability in vitro. To specifically target the CD133 (+) population, we encapsulated ATRA into nanoparticles conjugated with CD133 antibody using biodegradable poly(D,L-lactide-co-glycolide) (PLGA) polymer. The combination treatment significantly inhibited tumor growth compared to single drug treatments while the ATRA or SAHA monotherapy groups did not reduce or even promoted tumor growth. The combination of ATRA with SAHA specifically induces apoptosis of TICs. Targeting of TICs by NANOG antagonism increases drug susceptibility in tumor-bearing mice and achieves ∼90% tumor growth suppression. RNA-seq analysis identified that ATRA+SAHA treatment reduced expression of MicroRNA-22 (miR-22), leading to induction of PTEN and ten-eleven translocation enzymes (TET2), which demethylates DNA. PTEN induction promoted P-FOXO3A and induced BIM, leading to induction of apoptosis. The combined treatment Induced TET1/2 to demethylate p53-binding sites of NANOG promoter, leading to downregulation of NANOG. Taken together, combination treatment of ATRA with SAHA may serve as a novel avenue for HCC treatment. Novel combination of repurposed drugs is cost-effective therapeutic strategy to target microRNA for eradication of TICs, leading to inhibition of metastasis and recurrence. Citation Format: Chia-Lin Chen, Suresh Swaminathan, Ameya Kirtane, Jayanth Panyam, Keigo Machida. Selective inhibitors epigenetically modify and eradicate tumor-initiating stem-like cells through downregulating microRNA 22-mediated TET induction and apoptosis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4657.

Elevated expressions of fascin mRNA and protein in cancer cells have been correlated with aggressive clinical course, poor prognosis and shorter survival. Fascin is an evolutionarily conserved actin-binding protein that plays a key role in forming filopodia. It is reported that the lysine 471 residue (K471) of fascin can be acetylated and also involves in its actin binding, but the roles of K471 acetylation in filopodia formation are still uncertain. In this paper, we produced point mutants mimicking the active deacetylated (Lys471Arg, K471R) or inactive acetylated (Lys471Gln, K471Q) states of fascin. The actin-binding assay showed that the fascin K471Q mutation decreased the actin binding activity of fascin, compared with wild-type fascin (WT) and K471R mutant fascin in the esophageal cancer cells. Moreover, live imaging revealed that K471Q mutant fascin reduced the number and length of filopodia, whereas the K471R mutant increased filopodia frequency. The fascin K471Q mutation changed the straight filopodia became forniciform filopodia. The protrusion presented abnormal morphology when cells spread. Further functional experiments showed that the K471Q mutant fascin rendered the tumor cell migration, invasion and tumorigenesis decrease significantly, whereas K471R mutant fascin like WT fascin still maintained a strong ability. Taken together, we propose that the lysine K471 residue may be a key specific actin crosslinking site and also involved in the tumor biological function of fascin. Citation Format: Fa-Min Zeng, Xiao-Ning Wang, Ying-Li Zhang, Lian-Di Liao, En-Min Li, Li-Yan Xu. Acetylation of Fascin on the lysine 471 residue produces a change in growth and mobility phenotype of esophageal cancer cells by inducing F-actin rearrangement. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4424.

Several papers have implicated mutations in the AT-rich interactive domain containing protein 1B (ARID1B) in carcinoma formation in multiple tissue types, including the lung. The ARID1B protein functions as part of SWI/SNF, a multi-subunit chromatin remodeling complex that repositions nucleosomes and may act as a tumor suppressor in cancer. ARID1B is located in a region in 6q that was previously shown to have strong evidence of linkage with lung cancer risk. As it is one of the more promising candidates within the target region, this study presents the viability of ARID1B as a candidate gene for familial lung cancer. Here, we chose individuals from families in our previous linkage study that showed the strongest linkage signal on 6q. 75 individuals from 9 families were sequenced for 37Mb of chromosome 6 from 130Mb to 167Mb using Illumina technology and a custom Agilent kit. Genotypes were imported into GoldenHelix SVS 7 for quality control and dropped if read depth < 10, quality score < 10 or a quality score to read depth ratio < 0.5. Variants with more than 1 Mendelian error were dropped. Variants with a single Mendelian inconsistency were dropped only for that family. Two-point parametric linkage analysis at theta = 0 was performed using R and an implementation of the Elston-Stewart algorithm in the paramlink package using a penetrance model of 0.01/0.1/0.1 (dd/Dd/DD) and a disease allele (D) frequency of 0.01. Family 102 showed strong evidence for linkage near ARID1B. The highest LOD score (1.34) out of all sequenced variants was located near this gene in a known conserved intergenic region between ARID1B and NOX3. Four other LOD scores > 0.7 were found in the intergenic region between these two genes. Two are transcription factor binding sites; two are conserved genomic elements. We also observed two linked intronic variants of ARID1B in this family; an enhancer (LOD = 0.89) and a transcribed region (LOD = 0.73). Two other families (47 and 59) displayed evidence of linkage in ARID1B. Here, the interest is not the value of the LOD scores (top scores were 0.64 and 0.54), but the centering of the linked haplotype around the gene. Family 47 has 60 variants with LODs between 0.64 and 0.61; 45 located within ARID1B or known regulatory regions. Family 59 has 61 variants with LODs between 0.54 and 0.51; 29 located within ARID1B or known regulatory regions The variants appear to be spread evenly throughout ARID1B, suggesting the presence of linked haplotypes. Analyses are currently underway to reconstruct possible haplotypes in these families. The results of this study show that variants in the ARID1B gene at least partially account for the linkage signal on 6q in three of the nine most strongly linked families in our cohort and thus we believe that ARID1B is a plausible candidate gene for further studies involving familial lung cancer. Replication studies using 27 new extended families are currently underway to confirm this initial result. Citation Format: Anthony M. Musolf, Claire L. Simpson, Mariza de Andrade, Diptasri Mandal, Colette Gaba, Ping Yang, Ming You, Elena Y. Kupert, Marshall W. Anderson, Ann G. Schwartz, Susan M. Pinney, Christopher I. Amos, Joan E. Bailey-Wilson. Chromatin remodeling gene ARID1B is linked to familial lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2542.

MicroRNAs control the expression of various genes, and several miRNAs are aberrantly expressed in tumors. MiRNA-145 is markedly downregulated in various cancers including colon carcinoma, and in vitro studies have established its pro-apoptotic and anti-proliferative role. MiR-33a has only recently been connected to cancer through its capacity to downregulate the oncogenic kinase Pim-1. Despite the functional relevance of miRNAs suppressed in tumors, only a few studies have explored their therapeutic application. This miRNA replacement therapy is largely hampered by the lack of powerful non-viral delivery tools for their in vivo administration. In this paper, we establish, for the first time, miRNA replacement therapy by polyethylenimine (PEI)-mediated delivery of unmodified miRNAs. After systemic or local application of low molecular weight PEI/miRNA complexes, intact miRNA molecules are delivered into tumors, resulting in profound anti-tumor effects in s.c. colon carcinoma xenograft mouse models. Tumor inhibition is based on miR-145-mediated reduced proliferation, increased apoptosis and the concomitant repression of c-Myc and ERK5, which we identify for the first time as being regulated by miR-145. Furthermore, we introduce the systemic injection of PEI-complexed miR-33a as a novel therapeutic principle aiming at the in vivo downregulation of the oncogenic kinase Pim-1. Anti-tumor effects of the treatment with PEI/miRNA complexes are comparable to PEI/siRNA-mediated Pim-1 knockdown in vivo. Taken together, this is the first study to establish chemically unmodified miRNAs, complexed with PEI, as an efficient and biocompatible strategy for miRNA replacement therapy, and to introduce PEI/miR-145 and PEI/miR-33a complexes as anti-tumor compounds in colon carcinoma.

Glioblastoma (GBM), also named grade IV astrocytoma, is the most common and lethal malignant primary brain tumor. GBM is characterized by two main histopathological conditions: necrotic foci typically surrounded by areas of high cellularity and microvascular proliferation. The causes of such densely populated regions, known a “pseudopalisades”, remain poorly understood. Firstly, high cellularity was thought to be due to the rapid proliferation of GBM cells, however recent histological studies have shown that proliferation in pseudopalisading areas is significantly lower than in adjacent regions. Additionally, in pseudopalisades, apoptosis is substantially larger than in neighboring regions. These evidences suggest that pseudopalisades are due to other causes than simply higher proliferation or survival rates. Recent reports have pointed out that, in histological slices from GBM patients, more than 50% of pseudopalisades present clearly a central obstructed blood vessel and in more than 90% microscopic evidences of thrombosis are observed. As a consequence, oxygen and nutrients supply is compromised around the surrounding zones. It has been proposed that one of the driven forces of glioma aggressiveness is these nutrient and oxygen starvation. According to this hypothesis, GBM cell proliferation and secretion of pro-coagulant signals would cause thrombotic events, leading to hypoxia and nutrient depletion in the microenvironment. As a consequence, cells migrate towards nutrients and oxygen enriched regions, creating the characteristic GBM pseudopalisades. Eventually, these migrating cells would reach other blood vessels and eventually cause the collapse of these vessels, restarting the process and creating an expansive wave of tumor cells across the brain. This complex process is hardly reproducible using “in vitro” models because the conventional migration assays are unable to mimic the complex microenvironment described. Recently, microfabrication and microfluidic technologies have arisen as interesting alternatives for creating high-performance cell culture systems. In this paper we describe the design, fabrication and biological validation of a microfluidic device using SU-8 technology for three-dimensional GBM cell cultures under thrombotic conditions. The fabricated microdevice possesses a central microchamber to locate the cells embedded within a hydrogel, mimicking the ECM and allowing migration in three dimensions. On both sides of the microchamber, two lateral microchannels are filled with culture medium, allowing nutrient and oxygen diffusion towards the microchamber, mimicking the brain blood vessels. In this study we have demonstrated the GBM cells (U-251-MG) viability within the microdevice. Moreover, controlling medium flow through lateral microchannels we can mimic the GBM-associated thrombotic pathophysiological conditions. Under these thrombotic circumstances nutrient starvation leads to a chemotactic process and the formation of a migratory front similar to the pseudopalisades observed in vivo and validated with mathematical algorithms. Moreover, our results suggest that the whole process stimulates a more aggressive behavior of GBM cells. In an early stage when nutrients are plenty, GBM cells remain in a non-invasive phenotype. When nutrients are depleted, GBM cells initiate a migration process towards enriched regions, leading to the pseudopalisade formation. When compared with patient´s tumor pathology images, GBM cell invasion behavior follow the same pattern observed in vivo. This novel technique could help us to understand the mechanism of the pseudopalisade formation and to suggest novel therapeutic targets to avoid tumor progression. Besides, these microfluidic devices could represent an extremely useful platform to test new anticancer agents in a preclinical setting that mimics the complex GBM microenvironment. Citation Format: Jose M. Ayuso, Rosa Monge, Alicia Martínez-González, Guillermo A. Llamazares, Javier Berganzo, Aurelio Hernández-Laín, Jorge Santolaria, Manuel Doblaré, Pilar Sánchez-Gómez, Víctor M. Pérez-García, Ignacio Ochoa, Luis J. Fernández. An in vitro model for glioblastoma using microfluidics: Generating pseudopalisades on a chip. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr B04.

Growing evidence suggests that a small population of cells, called cancer stem cells (CSCs) contribute to the heterogeneity, recurrence and resistance to chemotherapy in breast cancer. In this regard, CSC is considered to be a good target to cure breast cancers. The extracellular signal-regulated kinase (ERK) signaling pathway is one of the major determinants in the control of diverse cellular process including the maintenance of CSCs. Kazinol-E is an antioxidant flavan purified from the root bark of Broussonetia kazinoki (Moraceae, paper mulberry). In this study, we found the anti CSC effect of Kazinol-E through targeting ERK pathway. It decreased the population of a breast cancer cell (MCF-7) CSCs which are CD44+/CD24- or aldehyde dehydrogenase positive, at the concentration that does not influence the growth of general MCF-7 cells cultured with serum. It targeted and inhibited ERK kinase activities in vivo in both CSC and in regular cultured cells suggesting the CSCs depend more than the rest of cancer cells do on Erk signaling for the survival. In vitro kinase assay using purified Erk-1 and p90RSK2, as the substrate, and the molecular docking study suggest that the Kazinol-E suppress the Erk activity by direct binding to the ATP binding pocket of Erk-1. Interestingly, a Kazinol-C which is very similar with Kazinol-E in structure did not show the Erk-1 inhibition nor anti CSC activity. These results collectively suggest that Kazinol-E may be a specific anti CSC agent targeting Erk pathway. Citation Format: Yu-Chae Jung, Seula Han, Li Hua, Hui-Yuan Zhao, Cheol-Jung Lee, Yong-Yeon Cho, Raok Jeon, Jae-Ha Ryu, Woo-Young Kim. Targeting cancer stem cell through blocking the Erk pathway with Kazinol-E from Broussonetia kazinoki. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2600. doi:10.1158/1538-7445.AM2015-2600

Aim: With the decreasing costs of genomics technologies, ever more data is being put into the public domain. Scientific papers only highlight a fraction of the information in the data, consequently further mining can answer drug discovery relevant questions and identify new targets. In this work we developed a bioinformatics pipeline, based on the collateral vulnerability hypothesis, to integrate several sources of public data and identify novel targets to form the basis of a new drug discovery project. Methods: Genomic data from TCGA was integrated with phenotypic data extracted from Mousemine, Flymine and Wormbase to identify loss-of-function aberrations in genes from families with essential predicted function. Follow-up experiments investigated the effect of siRNA knockdown of paralogs of genes of interest on various cellular phenotypes including proliferation, survival and senescence in gene deficient cell lines. A fragment screen was used to assess drugability of genes of interest. Results: The pipeline has been applied to several cancer types, and as a result a drug discovery project has been initiated against SMARCA2 in SMARCA4-deficient lung adenocarcinoma. SMARCA4 is a bromodomain-containing transcriptional co-activator within the multi-subunit SWF/SNF complex, which also possesses helicase and ATPase activities and functions to alter chromatin structure. SMARCA4-deficient cell lines harbour abrogating mutations, and previous studies have demonstrated that knockdown of SMARCA2, its functional paralog, in SMARCA4-deficient cell lines results in reduced cellular proliferation and survival. Moreover, SMARCA2 has been shown to be inactivated by epigenetic silencing in a proportion of human tumours. The collateral vulnerability hypothesis was tested in a panel of lung adenocarcinoma cell lines with SMARCA2- and/or SMARCA4-deficiencies. Experiments investigating the effect of siRNA knockdown confirmed both our hypothesis and the published data. A fragment screen against the bromodomain of SMARCA2 generated a high ‘ligandability’ index, suggesting that this target is druggable. Conclusion: SMARCA2 has been validated by our work and others as a target in SMARCA4 deficient lung adenocarcinoma. Future work will focus on elucidating the role of the bromodomain and the ATPase domain in SMARCA2/4 activity, and we are actively pursuing the identification of small molecule inhibitors of SMARCA2. An HTS has been undertaken against a library of >700 million compounds in a DNA-encoded library to identify novel hit matter that may ultimately be developed for therapeutic value. Citation Format: Phil Chapman, Nikki March, Graeme Thomson, Emma Fairweather, Samantha Fritzl, James Hitchin, Nicola Hamilton, Allan Jordan, Ian Waddell, Donald Ogilvie. Inhibition of SMARCA2: a novel target for SMARCA4-deficient lung adenocarcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5429. doi:10.1158/1538-7445.AM2015-5429

Background. Persistent infection with carcinogenic human papillomaviruses (HPV) is known to be cause of the majority of anogenital cancers as well as some head and neck cancers. With the progression of the disease, the HPV genome is commonly integrated within the host DNA, although the mechanism by which integration may promote the progression of the disease is not well-understood. We performed a systematic review of integration events in HPV-related cancers and analyzed the genomic regions where these events occur. Methods. A PubMed search was performed to collect all published data on integration loci. A total of 29 articles were eligible for the analysis. Determination of genomic integration sites for papers that provided exact nucleotide sequences of integration sites or viral fusion regions was done by using the Basic Local Alignment Search Tool (BLAST, http://blast.ncbi.nlm.nih.gov/Blast.cgi) for the GRCh37/hg19 build. We used the Encyclopedia of DNA Elements (ENCODE) data to investigate if certain genetic features clustered with integrations sites. The Wilcoxon rank sum test was computed to compare the observed and expected distributions of minimal distances from the integration sites with known nucleotide positions to the different genetic features. Results. As of November 2014, approximately 1,200 integration events were reported in the literature from both tumors and cell lines. The majority of integrations sites (53%) were reported in cervical tumors, 9% in head and neck tumors, 4% of oropharyngeal tumors and 23% in cell lines,. Six percent and 2% of the integration sites were found in high-grade and low-grade cervical lesions, respectively. The majority of reported integration events (77%) were related to HPV16. We found that integration sites from tumors were closer to known genes (p Conclusions. In summary, integration sites occur in genomic regions with enrichment for genetic elements, such as gene-rich regions, CpG sites, repetitive elements and open chromatin regions. This corroborates that accessible regions of the genome are preferentially targeted by integration and suggests that host genes may be functionally altered in development of HPV-related cancers. Citation Format: Clara Bodelon, Micahel Untereiner, Svetlana Vinokurova, Mitchell J. Machiela, Nicolas Wentzensen. Characterization of HPV integration sites in the human genome. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5580. doi:10.1158/1538-7445.AM2015-5580

Concomitant tumor resistance (CR) is the phenomenon according to which a tumor-bearing host inhibits the growth of secondary tumor implants. Ehrlich first described it in 1906, but this phenomenon remained forgotten for about 60 years. After its renascence, some groups have demonstrated that both immunogenic and non-immunogenic tumors can induce CR in different animal models. Metastases could be considered as secondary tumor implants developed spontaneously during the primary tumor growth, thus CR could be relevant for cancer progression. Clinical and experimental evidence suggest that the removal of human and murine tumors might be followed by an abrupt increase in metastatic growth, hence the primary tumor could exert a controlling action on its metastases. In previous papers we demonstrated that, in mice, two temporally separate peaks of CR can be detected during murine T-lymphoma (LB) primary tumor growth. The second peak of CR is mediated by most large-sized immunogenic and non-immunogenic tumors and is associated with the anti-tumor and anti-metastatic serum factor meta-tyrosine (m-tyr), an isomer of tyrosine not present in normal proteins. Based on this background, in this work we assessed whether CR was also occurring in human tumor experimental models. Athymic nude mice were inoculated s.c. in the right flank, with the human prostate cancer cell line PC3 (1 × 106, primary implant). After 14 days the animals received a second inoculation of PC3 cells in the left flank (1 × 106, secondary implant). The control group only received the secondary implant. The growth of the secondary implant was significantly reduced (92%; P Citation Format: Geraldine Gueron, Nicolás Anselmino, Damian Manchuca, Emiliano G. Ortiz, Maria Noelia Carabelos, Federico Schuster, Paula Chiarella, Alejandra Paez, Felipe M. Jaworski, Javier Cotignola, Roberto Meiss, Raul Ruggiero, Elba S. Vazquez. A second round for concomitant resistance in human cancer: A restraint upon metastasis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5199. doi:10.1158/1538-7445.AM2015-5199

Proteases exert control over cell behavior and affect many biological processes by making proteolytic modification of regulatory proteins. The purpose of this paper is to describe novel, important functions of matrix metalloproteinase (MMP)-26. α1-Antitrypsin (AAT) is a serpin, the primary function of which is to regulate the activity of neutrophil/leukocyte elastase. Insufficient antiprotease activity because of AAT deficiency in the lungs is a contributing factor to early-onset emphysema. We recently discovered that AAT is efficiently cleaved by a novel metalloproteinase, MMP-26, which exhibits an unconventional PH81CGVPD Cys switch motif and is autocatalytically activated in cells and tissues. An elevated expression of MMP-26 in macrophages and polymorphonuclear leukocytes supports the functional role of MMP-26 in the AAT cleavage and inflammation. We have demonstrated a direct functional link of MMP-26 expression with an estrogen dependency and confirmed the presence of the estrogen-response element in the MMP-26 promoter. Immunostaining of tumor cell lines and biopsy specimen microarrays confirmed the existence of the inverse correlations of MMP-26 and AAT in cells/tissues. An expression of MMP-26 in the estrogen-dependent neoplasms is likely to contribute to the inactivation of AAT, to the follow-up liberation of the Ser protease activity, and because of these biochemical events, to promote matrix destruction and malignant progression. In summary, we hypothesize that MMP-26, by cleaving and inactivating the AAT serpin, operates as a unique functional link that regulates a coordinated interplay between Ser and metalloproteinases in estrogen-dependent neoplasms.

Background: Using clinical samples and tumor cell lines, chromosomal aberrations and somatic mutations have been well explored in the field of pediatric oncology. However, genes susceptible to pediatric acute lymphoblastic leukemia (ALL) as well as lymphoblastic lymphoma (LBL) have not been delineated yet. Therefore, we examined copy number variations (CNVs) of genomic DNA by means of high-resolution comparative genomic hybridization (CGH) microarray to find significant susceptible genes to ALL and LBL. Methods: We investigated CNVs by array CGH in genomic DNA derived from a total of 58 children with ALL and LBL as case, and compared with control I (n=67): acute myeloid leukemia (AML)(n=10), other type of leukemia (n=4), non-LBL (n=7) and solid tumors such as neuroblastoma (n=46) as well as control II (N=301): adults patients with diabetic nephropathy and receiving dialysis (n=86), with neurological diseases (n=76) and with esophageal, gastric and colon cancers (n=72) plus control I (n=67). Normalization was performed using the Log2 signal ratio (sample DNA/control DNA) for individual 180K probes distributed throughout the whole genome. Control DNA was obtained from a single Japanese person. To compare data between two groups (e.g., ALL+LBL vs. control I or control II), an unpaired t test was used and subsequently corrected by the Benjamini-Hochberg false-discovery rate using GeneSpringGX v.11.5.1. Probes with a corrected p-value (=q-value) less than 0.05 and fold change greater than 1.8 or less than -1.8 were considered statistically significant. Significant gene was confirmed using real time polymerase chain reaction (PCR). Results: CNV of uridine diphospho glucuronosyltansferase 2B17 (UGT2B17) gene was statistically less deleted in children with ALL+LBL, compared with control I (q-value=5.51 x 10-12) and with control II (q-value=6.58 x 10-6). Presence or absence of UGT2B17 gene was reconfirmed by real time PCR. CNVs of other genes were similar in cases and controls. UGT2B17 glucuronidates several endogenous and exogenous compounds in particular steroid hormones as well as several pharmaceutical drugs such as anthraquinones and histone deacetylase inhibitor. As previously reported in some paper of adult solid tumors, CNV of UGT2B17 gene showed significant correlation to drug response. The frequency of CNV shows exceptional differences between populations from Africa, Europe, and Asia. Interindividual variability in UGT2B17 allele-frequency is accompanied by pronounced differences in gene expression characterized by more than 29 times higher mRNA levels in Caucasian compared with Japanese. Conclusion: These results suggest that homozygous or heterozygous gains of the UGT2B17 gene may be associated with a significant risk for pediatric ALL and LBL. Citation Format: Sae Ishimaru, Yuya Saito, Yuichi Yokokawa, Yuki Yuza, Takashi Kaneko, Mitsuyoshi Urashima. Uridine diphospho glucuronosyl-transferase 2B17 genotype and a risk of acute lymphoblastic leukemia and lymphoblastic lymphoma among Japanese children. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A9.

Glioblastoma multiforme is the most common and aggressive malignant primary brain tumour in humans, and has a very poor prognosis. As it is a well known fact that environmental factors especially radiation exposure is a major risk for glioblastoma multiforme and Iraq had been exposed in a relatively short period to 2 international wars with documented radiation exposure, so this paper might give justification for establishment of a new glioblastoma cell line. This cell line has been established from a cerebral glioblastoma multiforme in a 72-year-old Iraqi man who underwent surgery for an intracranial tumour in 2005. The morphology, growth kinetics, karyotype, immunocytochemistry and angiogenesis factor expression profile of this ANGM5 cell line were studied. The cell line has been grown continuously for more than 200 serial passages in standard culture media presenting no sign of cell senescence. The cultured cells are elongated or multipolar in shape and the population doubling time is 28 hours. The karyotype is complex and the chromosomal number varied between 46 and 48. The cell line is resistant to Temozolomide, cisplatin, vincristine and etoposide. This chemoresistance could be explained by the overexpression of the breast cancer resistance protein (BCRP) which found to be overexpressed by this cell line. Immunocytochemistry also showed that this cell line exhibited several characteristic features of glioblastomas, analysis of glial markers demonstrated that cells are positive for glial fibrillary acidic protein (GFAP), and negative for nestin and calbindin. Protein microarray analysis showed very high production of TIMP2 as well as other factors that are important for the invasiveness and aggressiveness of glioblastoma. This glioblastoma cell line is a useful addition to the cell lines currently available for study of the pathobiology and chemoresistance properties of glioblastoma multiforme as well as for antitumour drug discovery. Citation Format: Ahmed M. Al-Shammari, Ahmed A. Al-Juboory, Asmaa A. Al-Mukhtar, Amal M. Ali, Zaid A. Al-Hili, Nahi Y. Yaseen. Establishment and characterization of a chemoresistant glioblastoma cell line from an Iraqi patient. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1221. doi:10.1158/1538-7445.AM2014-1221

Ewing sarcoma (ES) is a highly malignant bone and soft tissue tumor with a 20% 5-year survival rate for patients with metastatic disease. Further, even clinically localized tumors are not successfully treated with local therapy like surgery and radiation, indicative of sub-clinical dissemination at presentation, as noted by James Ewing in his original papers. Extensive research has led to better understanding of the molecular pathogenesis of the disease but few discoveries have been successfully translated into improving treatment of ES patients. Our aim is to identify and characterize novel genomic features that can be developed into therapeutic targets for metastatic ES. This study documents the importance of one such ES-specific long non-coding RNA (lncRNA) in invasion and metastasis. Expression data from over a hundred ES primary tumors were compared to normal adult tissues as well as other types of pediatric and adult cancers to identify ES-specific coding and non-coding RNA transcripts. One highly expressed lncRNA, AK057037, was exclusively associated with ES. Further characterization of the transcript using loss of function studies showed that this 2.6 kb, 7 exon, polyadenylated RNA was directly regulated by EWS-FLI1, the chimeric oncoprotein that is the hallmark of ES and a major driver of tumorigenesis. In vitro, knockdown of the lncRNA resulted in reduced invasiveness of ES cells. In vivo, cells over-expressing the transcript showed statistically significant increase in metastasis compared to controls. Downstream targets included cell adhesion molecules such as cadherins and extracellular matrix proteins. Immunoprecipitation (IP) showed binding of the lncRNA to its direct targets. Binding of the lncRNA to other regulatory proteins also affected more global changes including promoter methylation and subsequent modulation of the downstream targets. We conclude that this lncRNA is a highly specific and unique ES biomarker. It acts as an oncogene by promoting invasion and metastasis of ES cells by controlling expression of genes involved in these pathways. Understanding this additional regulation by non-coding RNAs will enable development of multi-modal therapies that may prove to be more effective in treating aggressive disease. Citation Format: Sheetal A. Mitra, Anirban P. Mitra, Jonathan D. Buckley, William A. May, Philipp Kapranov, Robert J. Arceci, Timothy J. Triche. Long non-coding RNA promotes metastatic behavior in Ewing sarcoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4973. doi:10.1158/1538-7445.AM2014-4973

Protein phosphatase-6 (PP6) is a member of type-2A protein-Ser/Thr phosphatases, and reportedly regulates several cellular functions including DNA damage responses, NF-kB activation, and mitotic spindle formation. Additionally, some recent papers reported somatic mutations of PPP6c gene, encoding the catalytic subunit of PP6, in about 10 % of melanoma patients, suggesting its potential role as either driver or suppressor of melanoma development. In this study, we generated mice, in which the PPP6C gene is deleted in keratinocyte-specific manner (PP6-KKO). PP6-KKO mice were predisposed to formation of skin tumor upon DMBA-treatment. Our results demonstrate PPP6c has a tumor-suppressive function at least in skin tumorigenesis. Citation Format: Katsuhisa Hayashi, Honami Ogoh, Toshio Watanabe, Nobuhiro Tanuma, Hiroshi Shima. Promotion of skin tumorigenesis by deletion of Ppp6c gene. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5282. doi:10.1158/1538-7445.AM2014-5282

Colorectal cancer (CRC) is one of the leading causes of cancer death in both the Western world and Taiwan. Although CRC can be inherited, most CRCs are sporadic. The possible causes of colorectal cancer include a high fat diet, low fiber intake, obesity and sedentary lifestyle. A relatively limited number of oncogenes and tumor-suppressor genes_most prominently the APC, KRAS, and p53 genes are mutated in a sizeable fraction of CRCs. Even though numerous genes have been reported to be associated with tumorigenesis in CRC, only a few of them have been validated and used as biomarkers for predicting metastasis and treatment response in clinical applications. Our published paper showed that overexpression of high-mobility group AT-hook 2 (HMGA2) is associated with metastasis and unequivocally occurred in parallel with reduced survival rates of patients with CRC, and thus HMGA2 may potentially serve as a biomarker for predicting aggressive CRC with poor survivability and as an indicator of better response to radiotherapy. The Let-7a miRNA is a tumor suppressing miRNA that targets oncogenes, including HMGA2. Previous reports show that Let-7a miRNA may regulate miR-21 expression in cholesteatoma. The benign nature of cholesteatoma may be due to a balance between Let-7a and miR-21. The miR-21 has oncogene-like activity and is highly expressed in several types of cancers. Our present study also found that patients with high miR-21 expression had poor survival. In this study, we demonstrated the association of mir-21 and Let-7a in HMGA2 regulation in the pathogenesis of colorectal cancer and how the expression of these genes is associated with the clinical outcomes and radiotherapy response of CRC patients. Citation Format: Ya-Wen Cheng, I-Chien Chen. The role of mir-21 and Let-7a in HMGA2 associated colorectal cancer pathogenesis with different APC/k-ras status. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5246. doi:10.1158/1538-7445.AM2014-5246

Mieap, a p53-inducible protein, controls mitochondrial quality by repairing or eliminating unhealthy mitochondria via Mieap-induced accumulation of lysosome-like organelles within mitochondria (MALM) or Mieap-induced vacuole (MIV), respectively (1-4). BNIP3 and NIX are essential mediators of MALM. MIV generation is closely related to the endocytic pathway. However, the mechanism of MALM and MIV function and of their selection and regulation remains unclear. UVRAG was identified as a Mieap-interacting protein. UVRAG plays a role in canonical autophagy through the endocytic pathway. Interestingly, Mieap α, but not Mieap β, specifically interacts with UVRAG. Knockdown of UVRAG severely inhibited MIV generation induced by Mieap α. On the other hand, endogenous Mieap β, but not Mieap α, accumulated at and within mitochondria during the MALM process. These results, taken together, suggest that Mieap α has a specific role in MIV generation through an interaction with UVRAG by regulating the endocytic pathway. In addition, UVRAG and Mieap are inactivated in human cancers including colorectal cancer patients, suggesting that the MIV regulated by the interaction of Mieap and UVRAG may play a role in tumor suppression. In this paper, we discuss several possible mechanisms for the selection of MALM or MIV and the role of Mieap/UVRAG in tumorigenesis on the basis of our recent data. 1. Miyamoto Y et al. PLoS ONE 6: e16054, 2011 2. Kitamura N et al. PLoS ONE 6: e16060, 2011 3. Nakamura Y et al. PLoS ONE 7: e30767, 2012 4. Miyamoto T et al. Sci Rep 2: 379, 2012 Citation Format: Yasuyuki Nakamura, Hiroki Kamino, Yuri Saito, Noriaki Kitamura, Hitoya Sano, Hirofumi Arakawa. Specific role of Mieap α in Mieap-induced vacuole generation through an interaction with UVRAG. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 318. doi:10.1158/1538-7445.AM2014-318

More than 80% of cancer related deaths are caused by cancer cell metastasis, a crucial step in the onset of tumor development. Among the molecules involved in promoting cancer metastasis is the cell adhesion molecule CD44, whose role in promoting cancer cell motility and metastasis is well known. Despite this knowledge, the molecular mechanism through which CD44 promotes tumor development and cell metastasis is still unclear. CD146 (MUC 18) is another member of the cell adhesion molecule family, first identified in highly metastatic melanomas. The absence of CD146 in normal melanocytes and its high expression in melanomas suggests its tumor promoting actions. Despite the association between CD146 expression and development of melanoma, the expression patterns and the role of CD146 in normal and metastatic breast tissues are still controversial. In this paper we provided evidence clarifying some of these discrepancies by presenting CD146 as a negative downstream target for CD44. Finally, this study demonstrates a new role of CD44 in regulating neovascularization and in promoting cancer cell transmigration of blood vessels via regulation of its downstream target CD146. Citation Format: Mohamed E. Abdraboh, Hamad Al-Riyami, Yahya Al-Farsi, Allal Ouhtit, Andrew D. Hollenbach, Ishita Gupta, Somya Shanmuganathan, Madhwa Hg Raj. CD146, a suppressor of breast cancer, is a novel target of CD44-signaling. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3302. doi:10.1158/1538-7445.AM2014-3302

Background: Mitochondria are essential for energy conversation and cellular metabolism in eukaryotic cell. Mitochondria contain mitochondrial proteins that are encoded in the nucleus and synthesized in cytosol. Synthesized mitochondrial proteins are specially sorted by sub-mitochondrial compartment and then transported to their functional location. If mitochondrial proteins are not transported to their specific regions, it is reported that Mitochondria has a tendency to be implicated in various diseases including cancer. Mitochondria consist of mitochondrial outer membrane and inner membrane, especially, TOM complex are integrated in mitochondrial outer membrane. TOM40 is a central component of TOM complex has General import pore (GIP) to pass mitochondrial pre-protein. The goal of this study is to investigate the function of TOM40 in ovarian cancer. Methods: At first, we performed microarray to identify new target gene in ovarian cancer for nine human ovarian cancer cell line and seven human ovarian surface epithelial cell lines (HOSEs). We investigated target gene expression level in ovarian cancer cell line by RT-PCR and Western blot. To evaluate the association of target gene expression level and clinicopatholigical parameter in ovarian cancer, we carried out immunohistochemistry by using normal and ovarian cancer tissue. Second, we constructed stable DOV-13 and RMUG-S cell lines for overexpression and knock down of TOM40 protein using lentiviral system. We carried out proliferation, invasion and migration assay of each individual stable cell line. Results: We identified TOM40 as a new target gene for ovarian cancer by microarray data that was overexpressed in all nine human ovarian cancer cell lines than HOSEs. TOM40 protein level in borderline and cancer tissue was higher than normal tissue and benign tissue. TOM40 immuno-staining score of normal ovarian tissues is 1.23 (95% CI, 0.44-2.01) that compare with TOM40 immuno-staining score of benign, borderline, and epithelial ovarian tumors is 1.23 (95% CI, 0.44-2.01), 1.41 (95% CI, 0.45-2.37), 3.42 (95% CI, 2.65-4.20), and 5.04 (95% CI, 4.82-5.27), respectively. We understood TOM40 expression level of DOV-13 cells and RMUG-S cells is lower and higher than other ovarian cancer cell line. TOM40 overexpression in DOV-13 cell line inhibited the cell proliferation, invasion and migration than that of control cell line. TOM40 knock down in RMUG-S cell line induced cell proliferation, invasion and migration abilities than in control RMUG-S cell line. Conclusion: It is important to transport pathways of mitochondrial proteins, actually, the paper discussing directly association of TOM complex involving mitochondrial proteins transport and various diseases including cancer is not found. Our results suggest that TOM40 can be a role as putative tumor suppressor for ovarian cancer. Citation Format: Sol Kim, Hanbyoul Cho, Wookyeom Yang, Hyunja Kwon, Ha yeon Shin, Eunju Lee, Eun-Suk Kang, Jae-hoon Kim. Overexpression of TOM40 (translocase in the outer mitochondrial membrane 40) inhibits the cell proliferation, invasion and migration abilities in ovarian cancer cell lines. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2456. doi:10.1158/1538-7445.AM2014-2456

Recently, myeloid derived suppressor cells (MDSCs) have gained much attention due to their role in tumor immunity suppression as well as promotion of angiogenesis, tumor cell invasion, and metastases. However, the mechanisms that regulate MDSCs recruitment have not been well clarified. Here, we demonstrated that hypoxic tumor microenvironment stimulate the recruitment of MDSCs through increased production of macrophage migration inhibitory factor (MIF) and interleukin-6 (IL-6) by head and neck squamous cell carcinoma (HNSCC) cells. Knock-down (KD) of HIF-1/2α in HNSCC cells decreased MIF level but did not inhibit the MDSCs migration, because HIF-1/2α KD increased NF-κB activity which further enhanced IL-6 secretion. In vivo administration of neutralizing IL-6 antibody and inhibitors against MIF and NF-κB decreased MDSCs accumulation, accompanied by reduced micro-vessel density (MVD), lymphatic vessel density (LVD), and xenograft tumor growth. The supplementation of MDSCs increased MVD, LVD, and xenograft tumor growth. In conclusion, hypoxic HNSCC cells may stimulate the accumulation of MDSCs through secreting MIF and IL-6. This process is mediated by interactions between NF-κB and HIF-1/2α. Strategies targeting hypoxic tumor microenvironment may represent attractive approaches to HNSCC therapy. [G Zhu and Y Tang contributed equally to this paper.] Citation Format: Guiquan Zhu, Yaling Tang, Ling Li, Xinhua Liang. Tumor-derived macrophage Migration Inhibitory Factor and interleukin-6 cooperated in hypoxic accumulation of CD11b+Gr-1+ myeloid cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1547. doi:10.1158/1538-7445.AM2013-1547

The transcription factor nuclear factor-κB (NF-κB) is a key mediator of the inflammatory response, and its activation correlates with poor prognosis in non-small cell lung cancer patients. Oncogenic KRAS and epidermal growth factor receptor (EGFR) mutations occur frequently in lung adenocarcinoma and are associated with increased NF-κB activation in lung tumors. Recent papers have shown that inhibition of epithelial NF-κB signaling in a mouse model of Kras-mediated lung tumorigenesis decreases tumor formation. However, the mechanisms linking NF-κB signaling to tumor formation remain unclear. We hypothesize that epithelial NF-κB signaling is a common requirement for lung tumorigenesis. For our studies, we used mice that inducibly express oncogenic KrasG12D or EGFRL858R+T790M as well as a dominant negative inhibitor of κB signaling (DN-IκB) in Clara Cell Specific Protein (CCSP)-expressing lung epithelium (designated KrasDN or EGFRDN mice). EGFRDN mice developed smaller and fewer tumors after two months of doxycycline administration compared to mice expressing EGFRL858R+T790M alone, while KrasDN mice did not develop any tumors. In KrasDN mice, loss of Kras transgene expression and increased cell death were observed after one week of doxycycline, suggesting that NF-κB signaling may be required for survival of Kras mutant cells in vivo. In EGFRDN mice, NF-κB inhibition led to a significant reduction in the percentage of BAL neutrophils when compared to mice expressing EGFRL858R+T790M alone. In addition, NF-κB inhibition led to reduced expression of CXCR2 ligands CXCL1 and CXCL2, which are known NF-κB-regulated neutrophil chemoattractants. In vitro inhibition of CXCR2 did not affect proliferation of EGFR-mutant cells, suggesting that the critical function of CXCL1 and CXCL2 may be inflammatory cell recruitment/activation. Future studies will investigate the effects of CXCR2 inhibition and neutrophil depletion on EGFR-mediated tumor formation in vivo. Together, these results suggest that NF-κB signaling is required for both Kras- and EGFR-mediated lung tumorigenesis, but NF-κB's function in a tumor may depend on the driver mutation. From these studies, we hope to identify NF-κB-regulated genes critical for tumor formation as potential targets for the development of better therapeutic strategies for lung cancer patients. Citation Format: Jamie A. Ausborn, Taylor Sherrill, Rinat Zaynagetdinov, Vasiliy Polosukhin, Frank McMahon, Fiona Yull, Timothy Blackwell. Epithelial NF-κB signaling is required for different functions in Kras- and EGFR-mediated lung tumorigenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2873. doi:10.1158/1538-7445.AM2013-2873