bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2373: Assessing intra-tumor heterogeneity and tracking longitudinal and spatial clonal evolution by next-generation sequencing
Authors: Jiang Yuchao , Qiu Yu , Minn Andy J. , Zhang Nancy R.
Published: July 2016

Cancer is a disease driven by genetic and epigenetic alterations that follows Darwinian evolution. Recently, there have been increasing efforts to sequence the tumor from the same patient at multiple time points and/or from multiple spatially separated resections. Different snapshots of the same tumor have proved invaluable for identifying subclonal populations and inferring the tumor's history.

We propose a method, Canopy, for estimating the clonal history and for longitudinal and spatial comparison of mutation profiles from one or more samples derived from a single patient. Canopy accounts for normal cell contamination and reconstructs subclonal phylogeny utilizing both somatic copy number alterations (CNAs) and somatic single nucleotide alterations (SNAs). Canopy provides a general mathematical framework that enumerates all possible CNA-SNA phases and temporal orderings. Taking as input the mutant and reference allele frequencies for SNAs, and depth of coverage for CNAs, Canopy gives confidence assessments of all possible configurations of the cancer's clonal evolution.

Canopy is applied to three cancer sequencing datasets of varying study design, as well as to an extensive simulation study. On a whole-exome study of a transplantable metastasis model derived from human breast cancer cell line MDA-MB-231, Canopy successfully deconvolutes the mixed cell sublines, using the single cell sublines as ground truth, and identifies DNA signatures that can be prognostic of distant metastasis. On a whole-genome sequencing dataset of the primary tumor and relapse genome of a leukemia patient, Canopy predicts phylogenetic histories in concordance with existing knowledge. On a whole-genome sequencing dataset of the breast cancer tumor and its subsequent metastatic xenograft, Canopy's inferred clonal phylogeny is concordant with genomic markers of major clonal genotype and is confirmed by single-cell sequencing. Finally, through simulations, we explore the effects of various parameters on deconvolution accuracy, and evaluate performance with comparison against existing methods. Collectively, Canopy provides a rigorous foundation for statistical inference on repeated sequencing experiments from evolving populations delineated temporally and spatially.

Citation Format: Yuchao Jiang, Yu Qiu, Andy J. Minn, Nancy R. Zhang. Assessing intra-tumor heterogeneity and tracking longitudinal and spatial clonal evolution by next-generation sequencing. [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 2373.
Abstract 2381: A multiancestral model of colorectal cancer: in vivo evidence that early heterogeneity contributes to cancer progression
Authors: Leystra Alyssa A. , Luers Brook , Son Junbo , Sievers Chelsie K. , Wisinger Amanda M. , Schwartz Alexander R. , Zahm Christopher D. , Matkowskyj Kristina A. , Albrecht Dawn M. , Clipson Linda , Deming Dustin A. , Newton Michael A. , Halberg Richard B.
Published: July 2016

Background: Intratumoral heterogeneity has been linked to tumor progression and chemotherapy resistance in the clinic. Recent models of colorectal tumor evolution indicate that heterogeneity likely arises early during the first few cell divisions and is maintained in a non-Darwinian fashion with discrete clones coevolving. However, how this heterogeneity arises is not fully understood. We propose that some cancers are derived from multiple unique ancestors, and that discrete clones enhance establishment, growth, progression, and resistance to therapy.

Methods: Mice carrying the Min allele of Apc and expressing a constitutively active form of PI3K in a subset of colonic epithelial cells develop multiple adenomas and adenocarcinomas in the colon. Cell lineage tracing and fluorescent endoscopy were used to follow the progeny of individual founding cells through establishment, growth, progression, and response to targeted therapy. Cell sorting and 3D tumor spheroid co-culture were used to further examine the growth behavior and treatment response in vitro.

Results: Nearly half (44%; 30/68) of the tumors were derived from at least two ancestral clones. The presence of multiple clones was associated with an increased likelihood of a tumor becoming invasive (p = 0.006). Moreover, each clone was more invasive within multi-ancestral tumors than within their homotypic counterparts, indicating that the increased invasion is shared among clones rather than owing to a single dominant clone (p = 0.05). Additionally, the presence of both clones appeared to protect susceptible clones from targeted therapy. In vitro experiments demonstrated that co-evolved clones adopted similar growth patterns, whereas independently evolved clones did not.

Conclusions: Taken together, these data strongly indicate that distinct tumor founding cells and their coevolving progeny can contribute to tumor establishment and moreover can enhance growth and survival during tumor progression and response to therapy.

Citation Format: Alyssa A. Leystra, Brook Luers, Junbo Son, Chelsie K. Sievers, Amanda M. Wisinger, Alexander R. Schwartz, Christopher D. Zahm, Kristina A. Matkowskyj, Dawn M. Albrecht, Linda Clipson, Dustin A. Deming, Michael A. Newton, Richard B. Halberg. A multiancestral model of colorectal cancer: in vivo evidence that early heterogeneity contributes to cancer progression. [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 2381.
Abstract 239: Alteration of drug sensitivity according to the induction and reversion of epithelial-to-mesenchymal transition (EMT) in human lung adenocarcinoma cell lines harboring an EGFR mutation
Authors: Kurimoto Ryota , Iwasawa Shunichiro , Ebata Takahiro , Ishiwata Tsukasa , Sekine Ikuo , Tada Yuji , Tatsumi Koichiro , Koide Shuhei , Iwama Atsushi , Takiguchi Yuichi
Published: July 2016

Introduction: EMT phenotype is known to relate to drug resistance and immune tolerance of cancer. However, mechanism underling these phenomena, including the mechanism of the induction and reversion of EMT, are not fully understood.

Methods: EGFR mutated human lung adenocarcinoma cell lines, HCC-827 and PC-9, were treated with TGF-ß and/or FGF-2 to induce EMT. EMT was reversed by treating the induced EMT cells with either of PP242 (mTOR inhibitor), metformin and DMSO. The phenotypic alterations according to the induction and reversion of EMT were accessed by a wound healing assay for mobility, flow cytometry for cell cycle, MTT and apoptosis assays for drug sensitivity to cisplatin and gefitinib, and RT-PCR and fluorescence IHC for PD-L1 expression.

Results: The treatment with combination of TGF-ß/FGF-2 showed an efficient increase in expressions of mesenchymal markers and slug in both cell lines, and a decrease in E-cadherin expression in HCC827. In immunoblotting analyses, the Smad3 pathway in PC-9, and the Smad3, MEK/Erk and mTOR pathways in HCC-827 were involved in the induction of the EMT. The induced EMT was accompanied by enhanced cell motility and accumulation in the G0/G1 phases in both cell lines. The induced EMT also made the cells less sensitive to gefitinib in both cell lines, and to cisplatin in HCC-827. Increased PD-L1 expression was observed in both cell lines. Treatment of the induced EMT cells with PP242, metformin and DMSO reverted the altered expressions of epithelial and mesenchymal markers, cell motility and cell cycle. These agents reverted the EMT to different extents and through different pathways, depending on the cell lines. Reversion of the EMT using each of the 3 agents partly restored drug sensitivity, and suppressed PD-L1 expression.

Conclusion: Inducing EMT by treatment with TGF-ß/FGF-2 is an effective way to acquire drug resistance and up-regulation of PD-L1. Reversion of the EMT, therefore, has a potential to overcome drug resistance and immune tolerance of cancer.

Citation Format: Ryota Kurimoto, Shunichiro Iwasawa, Takahiro Ebata, Tsukasa Ishiwata, Ikuo Sekine, Yuji Tada, Koichiro Tatsumi, Shuhei Koide, Atsushi Iwama, Yuichi Takiguchi. Alteration of drug sensitivity according to the induction and reversion of epithelial-to-mesenchymal transition (EMT) in human lung adenocarcinoma cell lines harboring an EGFR mutation. [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 239.
Abstract 2393: Kidneys of VHL patients reveal the origin of renal clear cell carcinoma
Authors: Mubarak Mayyan , Sommaruga Samuel , Voigt David , Xu Xiacao , Kim Steve , Song Ailin , Vortmeyer Alexander O.
Published: July 2016

VHL disease is a classic tumor suppressor gene syndrome characterized by development of specific types of tumors in selective organs with nervous system and kidney being most consistently affected. Detailed studies on human spinal cord and cerebellum have previously revealed earliest stages of CNS tumorigenesis and the morphologic sequence resulting in development of frank tumors.

To elucidate earliest stages of renal clear cell carcinoma, we performed a similar approach in kidney tissues of four VHL patients and three sporadic control cases. From all cases, blocks of interest were procured, followed by serial sectioning and 3dimensional reconstruction of potential precursor lesions. The results reveal an abundance of foci with aberrant mesonephric clear cell proliferations that initially develop along the tubular lining, but have the potential to aggregate within individual tubules. This stage is followed by microscopic invasive clear cell aggregations which represent tumor precursor structures.

This study presents evidence for a consistent morphologic sequence for renal clear cell carcinogenesis. Molecular analysis of early steps within this sequence will allow for identification of earliest genetic and proteomic changes in the future. Therapeutic targeting of earliest changes may allow to develop preventive strategies for renal cancer development for VHL patients, potentially also for the non-VHL population.

Citation Format: Mayyan Mubarak, Samuel Sommaruga, David Voigt, Xiacao Xu, Steve Kim, Ailin Song, Alexander O. Vortmeyer. Kidneys of VHL patients reveal the origin of renal clear cell carcinoma. [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 2393.
Abstract 240: Functional significance of co-occurring mutations in PIK3CA and MAP3K1 in breast cancer
Authors: Avivar-Valderas Alvaro , McEwen Robert , Taheri-Ghahfarokhi Amir , Ahmed Ambar , Stetson Daniel , Dougherty Brian , Maresca Marcello , Hudson Kevin , Cruzalegui Francisco
Published: July 2016

PI3Kα signalling pathway is frequently hyper-activated in breast cancer (BrCa) with mutations of PIK3CA (∼45%) frequently found in luminal A BrCa samples. Genomic studies have also uncovered inactivating mutations in MAP3K1 (13-20%) and MAP2K4 (∼8%), two upstream kinases of the JNK apoptotic pathway in luminal A BrCa samples and co-occurring mutation of PIK3CA and MAP3K1 are found in ∼11% of PIK3CA mutants (TCGA). How these two alterations cooperate to promote tumorigenesis and impact the sensitivity to PI3Kα(AZD8835) and AKT (AZD5363) inhibitors is currently unknown. Using siRNA targeting and precise gene editing we have knocked down MAP3K1 in PIK3CA mutant cell lines to create in vitro models reflecting the mutational status of PIK3CA and MAP3K1 in BrCa patients. MAP3K1-deficient cells exhibited increased proliferation rate and decreased sensitivity to AZD8835 compared with parental control cells. In addition, mechanistic analysis revealed that siRNA mediated depletion of MAP3K1 enhances AKT phosphorylation and downstream signalling (i.e. pPRAS40, pFoxO1/FoxO3a) and provides resistance to AZD8835-mediated pathway inhibition. Using in vitro 3D-breast cancer organoid models we have found that MAP3K1 depletion increases overall acinar volume and counteracts AZD8835-mediated reduction of acinar growth. MAP3K1 deficient acini exhibited high pAKT levels in the presence of AZD8835 compared with control acini validating observations in 2D. Remarkably, whereas in control acini pAKT+ cells were restricted to the basal compartment, MAP3K1 deficient acini also exhibited pAKT+ cells in the luminal compartment suggesting anoikis resistance which is a hallmark in early stages of tumorigenesis. Elucidating the mechanistic basis for these clinically co-occurring mutations will provide insights into the mechanisms contributing to the oncogenic role of the PI3K pathway and future patient selection strategies.

Citation Format: Alvaro Avivar-Valderas, Robert McEwen, Amir Taheri-Ghahfarokhi, Ambar Ahmed, Daniel Stetson, Brian Dougherty, Marcello Maresca, Kevin Hudson, Francisco Cruzalegui. Functional significance of co-occurring mutations in PIK3CA and MAP3K1 in breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 240.
Abstract 2404: Dysregulation of lung developmental pathways associated with LKB1 loss in lung cancer
Authors: Kaufman Jacob M. , Lowe Cindy , Harris Bradford , Boyd Kelli , Amann Joseph , Eisenberg Rosana , Carbone David , Massion Pierre
Published: July 2016

Loss of the LKB1 tumor suppressor influences differentiation phenotypes in mouse models of lung adenocarcinoma and in human tumors. We utilize molecular data from resected human lung adenocarcinomas characterized by the TCGA to demonstrate that LKB1 deficient tumors exhibit a higher prevalence of neuroendocrine and gut-like differentiation markers than do LKB1 wild-type tumors (41% vs 9%, odds ratio = 7.3, P = 5.9E-19 for neuroendocrine differentiation; 36% vs 12%, odds ratio = 4.3, P = 2.5E-11 for gut-like differentiation. Fisher's exact test). Using copy number analysis and transcription factor enrichment analysis, we link these phenotypes to activation of particular transcription factors, namely FOXA2, HNF1A, ASCL1, and TTF1. We observe similar associations among non-small cell lung cancer cell lines, which may represent appropriate ideal models for further experimentation. Furthermore, we examine these differentiation phenotypes using immunohistochemical analysis of a tissue microarray (TMA), combined with RNAseq expression analysis, and MS/MS proteomic characterization of the same cohort of 36 tumors. Our molecular characterization confirms the neuroendocrine and gut-like differentiation phenotypes observed in the TCGA dataset and their association with LKB1-loss. Moreover, our immunohistochemical analysis demonstrates that these distinct differentiation subtypes are heterogeneously expressed within single tumors. Co-expression of neuroendocrine, gut-like, and respiratory differentiation is significantly more likely to occur within LKB1 deficient tumors, and is also associated with increased expression of a c-Kit positive sub-population that appears to lack expression of more differentiated markers. Among the TCGA tumors, this tri-lineage expression pattern is seen in 12.5% of LKB1-deficient tumors, compared to 1% of LKB1-wild-type tumors (Odds ratio 11.5, P = 3.2E-8. Fisher's exact test). These findings give further support to the role of LKB1 in regulating differentiation phenotypes in lung cancer, and may suggest a hierarchy of progenitor cells and lineage-specific subpopulations within these tumors.

Citation Format: Jacob M. Kaufman, Cindy Lowe, Bradford Harris, Kelli Boyd, Joseph Amann, Rosana Eisenberg, David Carbone, Pierre Massion. Dysregulation of lung developmental pathways associated with LKB1 loss in 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 2404.
Abstract 2412: Reconstructing intratumor heterogeneity: lessons from therapeutic intervention in patient-specific models
Authors: Schumacher Dirk , Boehnke Karsten , Keil Marlen , Silvestri Alessandra , Silvestrov Maxine , Hoffman Jens , Fichtner Iduna , Schaefer Reinhold , Regenbrecht Christian RA
Published: July 2016

Precision medicine approaches seeking to predict individual susceptibility to single or combinatorial drugs for advanced and/or metastasized solid cancers heavily depend on biological model systems that maintain key morphological, phenotypic and genomic features of the original tumor.

Here, we present a case study of a female patient with metastasized colorectal cancer. Using tissue from five distinct areas of the primary tumor and its metastasis of the synchronously removed primary tumor and liver metastasis, we generated cell cultures. To address cell morphology and phenotypic features we performed immunohistochemistry (IHC) and immunofluorescence imaging showing that these cell cultures express colon-specific marker proteins/combinations CDX2 and CK20+/CK7-. Ultra-deep panel sequencing of original tumor tissue samples revealed damaging mutations in KRAS (G12D), PIK3CA (H1047R) and TP53 (C242F). Two cell cultures showed an additional SNP in the tumor suppressor SMAD4 (R361H), which was not detected in the original tissue, thus hinting at the existence of a small sub-population of cancer cells with a divergent mutation pattern.

In parallel we established patient-derived xenografts (PDX) models from each region and investigated the response to targeted therapeutics, aiming at key molecules of the MAPK, PI3K/AKT and mTOR pathways in these genetically heterogeneous populations. drug response in single and mixed cell populations to address differential growth characteristics and response to mono- and combinatorial drug treatment. To distinguish the cells-of-origin and to calculate distributions/contribution of each population to the in vivo tumor growth in this model, sub-populations were color-coded using lentiviral constructs, expressing either a green or red tag. FACS analyses after treatment were performed and ratios of SMAD4mut(green) vs. SMAD4wt(red) were determined for each PDX tumor. These analyses, together with confirmatory IHC staining for the respective tag, showed significant effects on both tumor growth and population distributions in a treatment-dependent manner, advocating for systematic evaluation of combinatorial treatment in patients.

Citation Format: Dirk Schumacher, Karsten Boehnke, Marlen Keil, Alessandra Silvestri, Maxine Silvestrov, Jens Hoffman, Iduna Fichtner, Reinhold Schaefer, Christian RA Regenbrecht. Reconstructing intratumor heterogeneity: lessons from therapeutic intervention in patient-specific models. [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 2412.
Abstract 2433: Loss of chd5-mediated gene repression synergizes with MYCN to accelerate neuroblastoma tumorigenesis in zebrafish
Authors: Zimmerman Mark W. , He Shuning , Shin Jimann , Zhu Shizhen , Guo Feng , Mansour Marc , Reyon Deepak , Joung J Keith , Quan Jinhua , Yusufzai Timur , Look A Thomas
Published: July 2016

Neuroblastoma is a malignancy of the peripheral sympathetic nervous system (PSNS) and accounts for 10-15% of cancer deaths among children. For the 40% of patients presenting with high-risk disease, current therapeutic approaches are insufficient and long-term survival is less than 50%. Along with genomic amplification of the MYCN oncogene, hemizygous loss of the 1p36 chromosomal region is a major risk factor in neuroblastoma. The human CHD5 gene is a neuronal specific chromatin remodeling helicase that maps to 1p36, and is thus frequently lost in high-risk neuroblastoma. Our laboratory has previously generated a faithful model of pediatric neuroblastoma in the zebrafish driven by overexpression of the MYCN oncogene in the PSNS (dbh:MYCN). Additionally, zebrafish chd5 mutant alleles were created using the newly developed gene editing technologies TALEN and CRISPR-Cas9. The resulting chd5 mutant fish exhibit abnormal development of the PSNS in the form of expansion of the superior cervical ganglia and enlargement of the interrenal gland (adrenal medulla). Haploinsufficiency for Chd5 combined with dbh:MYCN expression accelerates the onset and increases the penetrance of neuroblastoma tumorigenesis in zebrafish, indicating a tumor suppressive function. Elevated p-ERK and PCNA+ cells in tumor tissue indicates that loss of Chd5, cooperates with MYCN overexpression to accelerate neuroblast proliferation in vivo. Chd5 (in addition to Chd3 and Chd4) is a core member of the epigenetic regulatory NuRD complex, which also contains HDAC1-2, MTA1-3, MBD2-3, GATAD2A/B and RBBP4/7. The conserved biological function of Chd5 is to silence gene expression through the maintenance of a repressed chromatin state. Tumors deficient for Chd5 expression exhibit reduced levels of the H3K27me3 histone modification, a marker of facultatively repressed genes. Future studies will further explore the mechanism and function of Chd5 so that the pathways mediating tumor suppression can be elucidated and that essential proteins in these pathways can be targeted in ways that exploit the synthetic lethal relationships that are established.

Citation Format: Mark W. Zimmerman, Shuning He, Jimann Shin, Shizhen Zhu, Feng Guo, Marc Mansour, Deepak Reyon, J Keith Joung, Jinhua Quan, Timur Yusufzai, A Thomas Look. Loss of chd5-mediated gene repression synergizes with MYCN to accelerate neuroblastoma tumorigenesis in zebrafish. [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 2433.
Abstract 2438: Genome-wide screen reveals a role for glucocorticoids in B cell development that can be exploited to improve treatment of B cell acute lymphoblastic leukemia
Authors: Pufall Miles , Kruth Karina , Tasian Sarah K.
Published: July 2016

Glucocorticoids (GCs) are a component of highly effective therapy for B cell acute lymphoblastic leukemia (B-ALL), the most common childhood cancer. Although successful in 90% of patients, the prognosis for the remaining 10% is dismal. Because sensitivity to GCs alone predicts outcome well, we hypothesized that enhancing or restoring GC sensitivity can improve outcome. To first determine how GCs kill B-ALL cells, we first measured the transcriptional response of sensitive B-ALL samples to dexamethasone (dex). In addition to apoptotic genes (BIM, BCL2), and metabolic genes (TXNIP) identified in other studies that contribute to cell death, we also found broad regulation of B cell development genes, including key checkpoints (ITGA4, IL7R, and BCL6). This suggested that high levels of GCs push arrested B-ALL cells through development, resulting in cell death. To validate these results, and to identify cellular pathways that modulate GC cytotoxicity, we used a next-generation shRNA screen to knock down every protein-coding gene in the genome. The screen identified 265 genes that significantly modulate GC sensitivity. In addition to validating the importance lymphoid development genes, the screen revealed which cofactors and chromatin modifiers collaborate with GR to orchestrate cell death gene programs. Further we were able to map critical signaling pathways with high resolution, including the pro-survival B cell receptor pathway. Inhibition of PI3Kδ at the apex of this pathway potentiated the response of GC-regulated genes to dex, and synnergized with dex to induce cell death in even the most refractory B-ALL tissue. These results indicate a previously unrecognized for GCs in B cell development that can be exploited to improve both the potency and treatment of lymhoid disease.

Citation Format: Miles Pufall, Karina Kruth, Sarah K. Tasian. Genome-wide screen reveals a role for glucocorticoids in B cell development that can be exploited to improve treatment of B cell acute lymphoblastic leukemia. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2438.
Abstract 2448: Suppression of MYCN-driven neuroblastoma malignant phenotype by telomerase-targeted tumor suppressor p53 transactivation
Authors: Tanimoto Terutaka , Tazawa Hiroshi , Noso Hiroshi , Oyama Takanori , Urata Yasuo , Kagawa Shunsuke , Noda Takuo , Fujiwara Toshiyoshi
Published: July 2016

Background: Neuroblastoma (NB) is a primary malignant tumor in the peripheral sympathetic nervous system. High-risk group of NB has an unfavorable clinical course, even after treatment with current chemotherapy regimens and aggressive surgical resection. Therefore, a novel therapeutic strategy is needed for the treatment of high-risk group of NB. Recent accumulating evidences in whole-genome sequencing analysis have shown that human telomerase reverse transcriptase (hTERT) gene rearrangement and MYCN gene amplification are predictive biomarkers for the high-risk group of NB, in which massive transcriptional activation of hTERT mRNA was frequently observed. We recently developed two types of hTERT-driven oncolytic adenoviruses, OBP-301 and OBP-702, in which the hTERT promoter drives the expression of the viral E1A and E1B genes for tumor-specific virus replication, and OBP-702 further expresses tumor suppressor p53 protein. In this study, we investigated whether OBP-301 and OBP-702 inhibit cell viability and MYCN protein expression in human NB cells with different MYCN status.

Methods: We used 3 human NB cell lines with or without MYCN amplification, including IMR-32 (MYCN-amplified), LA-N-5 (MYCN-amplified), SK-N-SH (MYCN-not amplified). The antitumor effects of OBP-301, OBP-702, and Ad-p53, a replication-defective adenovirus expressing p53 gene, were evaluated using XTT assay. Virus-induced apoptosis and MYCN suppression were analyzed by Western blot analysis.

Results: OBP-301, OBP-702, and Ad-p53 suppressed the viability of all 3 NB cell lines, whereas OBP-702 showed the most profound anti-tumor effect especially in MYCN-amplified NB cells. OBP-702 induced higher p53 expression than Ad-p53, resulting in the p53-mediated apoptotic cell death. Although MYCN expression in NB cells with MYCN amplification was downregulated after infection with all types of adenoviruses, the inhibitory effect of OBP-702 was the strongest among these adenoviruses.

Conclusions: These results suggest that a hTERT-driven oncolytic adenovirus OBP-702 is a promising antitumor agent to induce profound apoptotic cell death through p53 transactivation and MYCN suppression in human NB cells with or without MYCN amplification. In vivo experiments are under way to investigate the antitumor effect of OBP-702 against xenograft NB tumors.

Citation Format: Terutaka Tanimoto, Hiroshi Tazawa, Hiroshi Noso, Takanori Oyama, Yasuo Urata, Shunsuke Kagawa, Takuo Noda, Toshiyoshi Fujiwara. Suppression of MYCN-driven neuroblastoma malignant phenotype by telomerase-targeted tumor suppressor p53 transactivation. [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 2448.
Abstract 2452: Exosomes as mediators of paracrine signalling that promote invasive behavior in rhabdomyosarcoma
Authors: Ghayad Sandra E. , Rammal Ghina , Ghamloush Farah , Basma Hussein , Saab Raya
Published: July 2016

Exosomes are small membranous vesicles secreted into body fluids by multiple cell types, including tumor cells. Tumor exosomes contain intact and functional protein, mRNA and miRNA that may alter the cellular environment to favor tumor growth. Rhabdomyosarcoma (RMS) is an aggressive childhood soft tissue tumor, with two distinct subtypes, alveolar (ARMS) and embryonal (ERMS) histology. ARMS tumors are characterized in the majority of cases by a fusion oncoprotein PAX3-FOXO1, thought to be an initiating factor in tumorigenesis. To date, no studies have been performed to investigate the possible role or content of exosomes in paracrine signaling in RMS.

In the present study, we isolated and characterized exosomes secreted from RMS cells, using 3 ERMS (fusion oncoprotein negative) and 2 ARMS (fusion oncoprotein positive) cell lines. RNA analysis showed that small RNA were enriched in RMS-derived exosomes, therefore we focused on evaluating the miRNA content. Array expression analysis showed that exosome miRNA clustered together well, and to a higher extent than cellular miRNA, in ARMS cell lines. Similarly, in the 2 ERMS cell lines that had mutant p53, exosome miRNA clustered together and the pattern of expression was very different from the corresponding cellular miRNA. ARMS and ERMS exosome-enriched miRNA were different, and only 2 miRNA were found to be common among both; putative targets are implicated in cancer and inflammation. Functionally, using in vitro assays, we found that both ERMS- and ARMS-derived exosomes significantly increased the cellular migration and invasion of normal human fibroblasts, and had a positive effect on viability and proliferation of both fibroblasts and RMS cells. In vivo, Matrigel plug assay showed that RMS-derived exosomes had a positive effect on the migration and invasion of stromal cells, suggesting a positive role on angiogenesis.

We conclude that RMS-derived exosomes can exert specific paracrine effects on recipient cells, enhancing cell viability as well as invasive properties of fibroblasts and stromal cells, and therefore is likely to play a role in promoting RMS angiogenesis and metastasis. Ongoing studies are aimed at directly assessing the role of RMS-derived exosomes in clinical angiogenesis and metastasis, using in vivo murine models, in addition to dissecting the role of specific enriched miRNA on recipient cell biology.

Citation Format: Sandra E. Ghayad, Ghina Rammal, Farah Ghamloush, Hussein Basma, Raya Saab. Exosomes as mediators of paracrine signalling that promote invasive behavior in rhabdomyosarcoma. [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 2452.
Abstract 246: Role for ATP7A copper transporter in cisplatin resistance, tumorigenesis and metastasis
Authors: Shanbhag Vinit , Gudekar Nikita
Published: July 2016

Copper is a trace metal with a ready capacity to gain or donate electrons. This property is harnessed by numerous enzymes to perform vital functions in the body. In humans, copper is required for various biochemical processes including cellular respiration, connective tissue development, iron transport, and pigmentation. The same redox property which makes copper useful can also have deleterious effects if the proper balance is not maintained. Hence a delicate balance of copper should be maintained in the body. Cellular copper homeostasis is maintained by several different proteins. Ctr1 (copper transporter 1) has high affinity for copper and aids in copper uptake into the cell. ATP7A and ATP7B are copper exporting ATPases, which share 60% identity, are functionally homologous. Under low intracellular copper concentrations, ATP7A is localized to the TGN (trans-Golgi network), where it transports copper to newly synthesized cuproenzymes. Under elevated copper concentrations, ATP7A traffics to post TGN vesicles and to the plasma membrane, subsequently releasing the copper load by fusion with the plasma membrane.

There are emerging studies implicating a role of ATP7A in resistance against the platinum-based chemotherapy agent, cisplatin, which is widely used to treat cancer. Previous studies have correlated ATP7A expression to the sensitivity of cultured cells to cisplatin. Here, we report that deletion of the Atp7a gene in isogenic RAS-transformed mouse embryonic fibroblast (MEF) cells not only confers sensitivity to cisplatin, but markedly prevents tumor formation in the absence of chemotherapy agent. Deletion of Atp7a in the breast cancer cell line, 4T1, was also found to suppress tumorigenesis and markedly reduce metastasis to the lungs. The ATP7A knockout 4T1 cells showed a significant decrease in Lysyl Oxidase (LOX) activity as compared to the 4T1WT cells. Given the numerous studies showing that LOX family involved in breast tumor metastasis, ATP7A might play a role in tumor progression by virtue of its requirement for metallating LOX. The ATP7A knockout 4T1 cells exhibited reduced survival as compared to the 4T1WT cells in the presence of physiological concentrations of hydrogen peroxide or hypoxia, suggesting that hyper-accumulation of copper sensitizes these cells to ROS production. Taken together, these findings identify the ATP7A copper transporter at the nexus of platinum-drug resistance, tumorigenesis and metastatic pathways, underscoring its potential as a therapeutic drug target at multiple stages of carcinogenesis.

Citation Format: Vinit Shanbhag, Nikita Gudekar. Role for ATP7A copper transporter in cisplatin resistance, tumorigenesis and metastasis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 246.
Abstract 2464: Disrupting the epigenetic modifier HMGA2 in DIPG and GBM inhibits tumor invasion, growth and tumorigenicity
Authors: Kaur Harpreet , Hütt-Cabezas Marianne , Taylor Isabella , Weingart Melanie , Rodriguez Fausto , Eberhart Charles G. , Raabe Eric H.
Published: July 2016

Diffuse intrinsic pontine glioma (DIPG) and glioblastoma (GBM) are lethal, incurable brain tumors with poor prognosis. Identifying and targeting novel molecular markers regulating tumor invasion and growth can lead to development of better therapies. HMGA2, a minor-groove DNA-binding protein, is a transcriptional modulator in normal and cancer stem cells. We hypothesized that HMGA2 contributes to pediatric high grade glioma invasion and tumorigenicity through its ability to alter the transcription of large numbers of genes. We found increased expression of HMGA2 in a subset of GBM and DIPG tumors by western blotting and immunohistochemistry. We confirmed this result in multiple patient-derived tumor cell lines, including 3 DIPG neurosphere cell lines. Lentiviral short hairpin RNA (shRNA)-mediated reduction of HMGA2 in GBM cells significantly reduced invasion in transwell assay and colony formation in soft agar assay (shHMGA2 vs. shControl, P<0.01). Similarly, shRNA mediated suppression of HMGA2 in DIPG cell lines reduced proliferation as measured by BrdU incorporation, reduced invasion by transwell assay and increased apoptosis by cleaved caspase-3 (CC-3) immunofluorescence (shHMGA2 vs. shControl, P<0.01). Pharmacological inhibition of HMGA proteins using the DNA minor-groove binding drug Netropsin significantly inhibited growth (BrdU assay) and increased apoptosis (CC-3 immunofluorescence) of DIPG cell lines (P<0.01). Mice bearing orthotopic GBM xenograft cell lines transduced with HMGA2 shRNA lived longer (median survival = 108 days) compared to control shRNA (67.5 days, P<0.0001 by log-rank analysis). Our results suggest an oncogenic role of HMGA2 in promoting DIPG and GBM invasion and growth, identifying HMGA2 as a potential therapeutic target in these devastating tumors. Netropsin or other minor groove binding agents may be effective therapeutics for pediatric high-grade gliomas.

Citation Format: Harpreet Kaur, Marianne Hütt-Cabezas, Isabella Taylor, Melanie Weingart, Fausto Rodriguez, Charles G. Eberhart, Eric H. Raabe. Disrupting the epigenetic modifier HMGA2 in DIPG and GBM inhibits tumor invasion, growth and tumorigenicity. [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 2464.
Abstract 2468: Combined targeting of the EWS/ETS transcriptional program by BET bromodomain and PI3K pathway inhibition blocks tumorigenicity and increases apoptosis in Ewing sarcoma
Authors: Hensel Tim , Giorgi Chiara , Becker-Dettling Fiona , Calzada-Wack Julia , Neff Frauke , Buch Thorsten , Schmidt Oxana , Schäfer Beat W. , Burdach Stefan , Richter Günther HS
Published: July 2016

Ewing sarcomas (ES) are highly malignant bone or soft tissue tumors. Genetically, ES are defined by balanced chromosomal EWS/ETS translocations that give rise to chimeric proteins (EWS-ETS), which generate an oncogenic transcriptional program associated with altered epigenetic marks throughout the genome.

By use of an inhibitor (JQ1) blocking BET bromodomain binding proteins (BRDs) we strikingly observed a strong down-regulation of the predominant EWS-ETS protein EWS-FLI1 in a dose dependent manner. This was further enhanced by co-treatment with an inhibitor (BEZ235) of the PI3K pathway. Microarray analysis revealed JQ1 treatment to block a typical ES associated expression program. The effect on this expression program was mimicked by RNA interference of BRD3 or BRD4 expression but not by BRD2 blockade, indicating that the EWS-FLI1 mediated expression profile is at least in part mediated via such epigenetic readers.

JQ1 treatment not only suppressed an ES specific expression profile but also blocked contact dependent and independent proliferation of different ES lines. But subsequent analysis of proliferation after transient knockdown of either BRD3 or BRD4 did not recapitulate inhibition of proliferation as observed after JQ1 treatment, indicating the necessary simultaneous blockade of both proteins. However, inhibition of proliferation after JQ1 treatment was due to a partial G1 arrest and S phase elongation of the cell cycle. In addition, induction of apoptosis as demonstrated by PARP1-, CASP7-cleavage and increased CASP3 activity significantly contributed to the reduction of the proliferative ability of ES lines. Single or combination treatment with the PI3K/mTOR inhibitor BEZ235 increased apoptosis of ES cell lines although single treatment with BEZ235 was less effective than JQ1 application. Consequently, tumor development was dose dependently suppressed with increased formation of apoptotic bodies in a xeno-transplant model in immune deficient mice, overall indicating that ES may be susceptible to treatment with epigenetic inhibitors blocking BET bromodomain activity and the associated pathognomonic EWS-ETS transcriptional program.

Citation Format: Tim Hensel, Chiara Giorgi, Fiona Becker-Dettling, Julia Calzada-Wack, Frauke Neff, Thorsten Buch, Oxana Schmidt, Beat W. Schäfer, Stefan Burdach, Günther HS Richter. Combined targeting of the EWS/ETS transcriptional program by BET bromodomain and PI3K pathway inhibition blocks tumorigenicity and increases apoptosis in Ewing sarcoma. [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 2468.
Abstract 2469: The role of L1 in proliferation and chemoresistance of retinoblastoma
Authors: Jo Dong Hyun , Lee Kyungmin , Kim Jin Hyoung , Jun Hyoung Oh , Kim Young Hoon , Cho Young-Lai , Yu Young Suk , Min Jeong-Ki , Kim Jeong Hun
Published: July 2016

Retinoblastoma is the most common intraocular malignant tumor in children, affecting approximately 1 in 20,000 live births. In this study, we investigated the role of L1, a transmembrane protein, in proliferation and chemoresistance of retinoblastoma to figure out the potential for the novel therapeutic target. Retinoblastoma tissues demonstrated varying degrees of L1 positivity in 86.6% (26/30) of samples. In particular, the degree of L1 positivity was inversely related with that of Flexner-Wintersteiner rosette formation. Further in vitro studies using stable cell lines which showed up- or down-regulation of L1 demonstrated that L1 was associated with cell-cell adhesion and proliferation of retinoblastoma cells. Retinoblastoma cells with low expression of L1 showed decreased tumor formation in vivo. In addition, L1 expression was related with resistance to carboplatin, one of the most-widely utilized chemotherapeutic agents against retinoblastoma. In line with these results, retinoblastoma cells with higher expression of L1 demonstrated increased resistance to carboplatin in vivo. We also observed diffuse and dense expression of L1 in retinoblastoma tissues from 4 patients who underwent enucleation despite intensive chemotherapy based on carboplatin. Taken together, L1 was related with proliferation and chemoresistance of retinoblastoma and might be a potential therapeutic target of retinoblastoma.

Citation Format: Dong Hyun Jo, Kyungmin Lee, Jin Hyoung Kim, Hyoung Oh Jun, Young Hoon Kim, Young-Lai Cho, Young Suk Yu, Jeong-Ki Min, Jeong Hun Kim. The role of L1 in proliferation and chemoresistance of retinoblastoma. [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 2469.
Abstract 2470: Isolation and characterization of cancer stem cells from the human medulloblastoma cell line DAOY: adherent vs. suspension in vitro cell cultures
Authors: de la Rosa Javier , Sáenz Antoñanzas Ander , Fan Xing , Meléndez Bárbara , Rey Juan A. , Castresana Javier S.
Published: July 2016

Brain tumor stem cells (BTSC) in vitro cultures have been made possible by the formation of neurospheres in suspension conditions with the use of serum free medium supplemented with specific growth factors, as B27, EGF, or b-FGF. In this work, we tested a more recent alternative method for the culture of BTSC, using laminin pre-coated flasks and the same medium used for the neurosphere growth, which leads to BTSC expansion attached to a layer.

BTSC cultures in adherence conditions by the use of laminin can afford a more homogeneous exposition to growth factors than sphere cultures do, which promotes a higher enrichment in stem-like cells. Also, it is known that the laminin interactions with integrines or other components of the cell matrix can lead to inhibition signals of cell differentiation. Moreover, the monolayer conditions can support a higher number of cells in the flask.

The medulloblastoma cell line DAOY was cultured in different conditions of normoxia and hypoxia, and several cell populations were obtained for in vitro and molecular experiments.

An increase in the stem features was shown when cells were successively cultured in hypoxia conditions. By contrast, a reduction in these properties was appreciated when cells were exposed to differentiation. In addition, the CD133+ and CD133- subpopulations were isolated from cells grown in laminin pre-coated flasks, and in vitro experiments showed that the CD133+ fraction represented the stem population and it could have BTSC with a higher probability than the CD133- fraction.

The colony formation assay presented similar results for the two cultured methods, but clonogenicity in soft agar was higher in the laminin culture cells. In the cell migration assay, medullospheres migrated similarly to the laminin cultured cells, since both cell populations lost their adherence capacity after being cultured with serum free medium. Gene expression of CD133, CD15 and EZH2 was compared as well, and significant differences were not detected between the two cell populations.

We can conclude that the laminin culture method in adherent conditions and the medullosphere traditional culturing method in suspension are similarly good for obtaining BTSC in DAOY medulloblastoma cell line.

Citation Format: Javier de la Rosa, Ander Sáenz Antoñanzas, Xing Fan, Bárbara Meléndez, Juan A. Rey, Javier S. Castresana. Isolation and characterization of cancer stem cells from the human medulloblastoma cell line DAOY: adherent vs. suspension in vitro cell cultures. [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 2470.
Abstract 248: Metformin sensitizes endometrial cancer cells to chemotherapy by inhibiting IDH1-TET1 pathway
Authors: Bai Mingzhu , Zhang Zhenbo , Yu Yinhua , Feng Youji
Published: July 2016

Chemotherapy-resistance is the major obstacle for endometrial carcinoma patient administration. Accumulated evidences indicate that metformin sensitizes cancers from several different sites to chemotherapy drugs and reverses progestin resistance in endometrial cancer. Our group has demonstrated that metformin reverses progestin resistance and enhances the rate of cell-killing induced by chemotherapeutic agents in endometrial cancer cells by downregulating GloI expression. Recent study has revealed that aberrant expression of IDH1 contribute to chemotherapy resistance. However, the role of IDH1 in metformin induced chemotherapy sensitivity is not well characterized. As a critical enzyme of the tricarboxylic acid (TCA) cycle, IDH1 catalyzes isocitrate to produce α-ketoglutarate (α-KG), a substrate of TET1, which control TET1-mediated epigenetic modulation of the target genes, including the chemotherapy-resistant genes. In the present study, we have found that IDH1 is overexpressed in endometrial cancers compared with benign endometrial lesions and normal endometria by immunohistochemical analysis of specimens (200 cases of endometrial cancers, 10 cases of benign endometrial lesions and 10 cases of normal endometria). Tissue microarray immunohistochemical detection showed the expression of IDH1, TET1 and 5hmc was positively correlated in different pathological types of endometrial tissue. Interestingly, we found that metformin reduces expression of IDH1 at protein levels with a time- and dose-manners in endometrial cancer cells. Furthermore, overexpression of IDH1 increased TET1 at protein level and blocked metformin-sensitized endometrial cancer to chemotherapy drugs, whereas decrease IDH1 expression lessened TET1 expression and paralleled with the attenuated chemotherapy-resistance. Mechanistic investigations revealed that TET1 involved in metformin-enhanced chemotherapy sensitivity via IDH1 by western blotting and DNA dot blotting assays, this may attribute to that reduced production of α-KG resulted from metformin-suppressing IDH1 is not enough to start TET1-mediated epigenetic modulation of chemotherapy resistant genes. Overall, our findings demonstrate that IDH1 regulates α-KG level and TET1 expression, which in turn affecting metformin mediated endometrial cancer chemotherapeutic sensitivity. Our study suggests IDH1 is a potential target to overcome cisplatin and paclitaxel resistance in endometrial cancer.

Citation Format: Mingzhu Bai, Zhenbo Zhang, Yinhua Yu, Youji Feng. Metformin sensitizes endometrial cancer cells to chemotherapy by inhibiting IDH1-TET1 pathway. [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 248.