bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2421: Circulating tumor cells reveal the genetic evolution of metastatic breast cancer
Authors: Joosse Simon A. , Babayan Anna , Prieske Katharina , Indenbirken Daniela , Alawi Malik , Burandt Eike C. , Grundhoff Adam , Müller Volkmar , Pantel Klaus
Published: July 2016

BACKGROUND. Anti-cancer treatment failure is caused by the genetic and phenotypic heterogeneous properties of the disease. Early dissemination of circulating tumor cells (CTCs) into the blood circulation of cancer patients allows for parallel genetic evolution of the primary tumor and metastases. This study investigated the genomic make-up of CTCs originating from metastasis as a so called “liquid biopsy” and compared these with the different clones of the archived autologous primary tumor on single cell level.

METHODS. From two breast cancer patients, individual CTCs were isolated from blood using Ficoll density gradient followed by micromanipulation of keratin positive cells. Single cells from archived primary breast tumors from the same patients were captured by laser microdissection. DNA was isolated and amplified by whole genome amplification and copy number alterations (CNA) were obtained by shallow, whole genome next generations sequencing (NGS).

RESULTS. From the first breast cancer patient, the genomes from 50 single cells from the primary tumor were sequenced. An unsupervised phylogenetic cluster analysis based on CNA revealed five distinct subclones that displayed increasing chromosomal instability from the first to the last cluster. Using support vector machine (SVM) learning, the CNA profiles of 42 CTCs were residing mostly to the first three clones with few chromosomal aberrations, whereas only one CTC resided to the fourth clone and none to the last with many chromosomal aberrations. The tumor from a second breast cancer patient displayed the presence of three genetically distinct clones with increasing chromosomal instability after sequencing 11 single cells. CTCs (n = 12) from this patient at metastatic disease were classified to the last branch using SVM, however with low probability. After repeating unsupervised clustering on the genomes of the primary tumor tissue and CTCs, a fourth branch was formed with CTCs only.

CONCLUSION. Our results suggest that therapy resistant metastases in breast cancer patients can originate from tumor clones from early stages of tumor evolution and may genetically still be similar (patient 1). On the other hand, further genetic progression may also take place (patient 2) after which the genetic landscape of the metastasis does not resemble the primary tumor anymore. These results underline the importance of “liquid biopsy” in the diagnosis of metastatic cancer.

Citation Format: Simon A. Joosse, Anna Babayan, Katharina Prieske, Daniela Indenbirken, Malik Alawi, Eike C. Burandt, Adam Grundhoff, Volkmar Müller, Klaus Pantel. Circulating tumor cells reveal the genetic evolution of metastatic 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 2421.
Abstract 2423: Deep phenotyping of dissociated solid tumor cells from breast cancer specimens
Authors: Middlebrook Aaron J. , Llontop Peter , Hanley Mary Beth , Porter Warren , Hahn Friedrich , Snowden Eileen , Blaesius Rainer , Ghanekar Smita
Published: July 2016

Exploring tumor heterogeneity with the goal of improving outcome has led to the need to glean as much information as possible at an individual cell level from these valuable specimens. Traditional approaches to solid tumor analyses fail to reveal the diverse range of cellular compartments beyond tumor cells that comprise the tumor microenvironment. A comprehensive approach to tumor interrogation requires efficient tissue dissociation to facilitate analysis at the single-cell level. Compared to current methods, single-cell analysis of tumor derived cell suspensions by flow cytometry has the potential to provide a more thorough understanding of the many subpopulations within the tumor microenvironment and the cell-to-cell interactions that govern this space.

Here, we demonstrate an efficient workflow that enables comprehensive cell analysis of solid tumors from breast cancers. After dissociating human breast cancer biopsies from primary tumors into cell suspension, we analyzed the immune compartment and the cancer/stromal cell compartment by multicolor flow cytometry, using 26 markers. A comprehensive analysis of this data set reveals the heterogeneity within the tumor microenvironment on a phenotypic level, which might have potentially significant correlations to the clinical status and molecular phenotype of the cancer. These results encourage the expansion of the use of flow cytometry as a means of solid tumor biopsy analysis, highlighting the potential clinical value of this approach in disease management.

Citation Format: Aaron J. Middlebrook, Peter Llontop, Mary Beth Hanley, Warren Porter, Friedrich Hahn, Eileen Snowden, Rainer Blaesius, Smita Ghanekar. Deep phenotyping of dissociated solid tumor cells from breast cancer specimens. [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 2423.
Abstract 2426: Detection of somatic mutations associated with pulmonary metastasis of osteosarcoma by whole exome sequencing
Authors: Iwata Shintaro , Tatsumi Yasutoshi , Yonemoto Tsukasa , Kamoda Hiroto , Ishii Takeshi , Nagase Hiroki , Ohira Miki
Published: July 2016

Objective: Pulmonary metastasis is a main cause of death for the patients with osteosarcoma (OS), although no specific genetic alteration associated with pulmonary metastasis has been reported. The objective of this study was to identify candidate somatic genetic alteration contributed to pulmonary metastasis in pediatric OS.

Methods: Five sample sets including the tissues taken from primary tumor (P), resected pulmonary metastatic tumor (M), and normal tissue (N) of the same pediatric OS patients were analyzed by whole exome sequencing with Ion Torrent Proton sequencer. 40ng of genomic DNAs extracted from each tissue were used for multiplex PCR amplification with Ion Ampliseq Exome Kit. Data analysis including alignment to the hg19 human reference genome and variant calling was done using the Torrent Suite Software. Obtained genomic data was validated by visualizing in Integrative Genomics Viewer or capillary sequencing. Candidate somatic genetic alterations were annotated by wANNOVAR database. Pathway analysis was performed using GeneMANIA.

Results: Mean read depths on the P, M, and N were 219, 212, and 109, respectively. We identified 1364 and 1311 candidate non-synonymous somatic single nucleotide variants (SNVs) among the P and M, respectively. After the validation and filtering, we detected 135 non-synonymous SNVs within the exon region in M. Among these, 32 SNVs were overlapped with those in P. Only 2 recurrent SNVs were identified within the whole SNVs. C:G>T:A transition mutations were frequently observed in both P and M, and the frequency were higher in M comparing to P. Pathway analysis showed a significant enrichment of the candidate SNVs in genes involved in extracellular matrix regulation (FDR;2.37E-03).

Conclusion: We have identified 137 SNVs associated with pulmonary metastasis from pediatric OS. Extracellular matrix-relating signature might contribute to the genesis of pulmonary metastasis.

Citation Format: Shintaro Iwata, Yasutoshi Tatsumi, Tsukasa Yonemoto, Hiroto Kamoda, Takeshi Ishii, Hiroki Nagase, Miki Ohira. Detection of somatic mutations associated with pulmonary metastasis of osteosarcoma by whole exome 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 2426.
Abstract 2435: Amplification of CDK4 and MDM2 is associated with atypical clinical features in high risk neuroblastoma patients
Authors: Fransson Susanne M. , Kryh Hanna , Javanmardi Niloufar , Ambros Inge M. , Berbegall Ana P. , Ora Ingrid , Noguera Rosa , Asmundsson Jurate , Sandstedt Bengt , Ambros Peter F. , Kogner Per , Martinsson Tommy
Published: July 2016

MYCN-amplification and 11q-deletion are important, although incomplete, markers of high-risk neuroblastoma. Thus, characterization of additional genomic alterations that can be used as prognostic and/or predictive markers is of clinical importance in order to provide best treatment possible.

By using SNP-microarrays we identified a small group of neuroblastomas with high grade amplification of one or multiple loci on 12q, commonly involving the potential oncogenic target genes CKD4 (12q13-14) and/or MDM2 (12q15). The CDK4 and MDM2 regions were co-amplified in 13/16 samples, two tumors had CDK4-amplification in absence of MDM2-amplification while one tumor had MDM2-amplification without CDK4-amplification. Exome sequencing was performed on seven tumors and whole genome sequencing (WGS) was performed on tumor and constitutional DNA from one patient with 12q-amplification. No novel protein altering SNVs were detected within the amplified regions except a rare INHBE mutation in one patient. The tumor examined with WGS show high degree of structural rearrangements including chromothriptic features on chromosome 12 leading to high grade amplification of CDK4 and MDM2. This sample displayed 21 somatic protein changing alterations although not in any gene with known function in chromatin stability or DNA repair.

Interestingly, the majority of the 12q-amplified neuroblastomas were of abdominal origin, some with renal location with initial suspicion of Wilms’ tumor. Atypical metastatic pattern were also seen in this patient group showing low degree of bone marrow involvement favoring other metastatic sites such as lung.

The consistent co-amplification of two separate chromosome 12 regions in this subset of neuroblastoma suggests that there are one or more genes with importance in tumor development/progression. Our study indicates that CDK4 appears as main target in this 12q-amplified neuroblastoma subgroup although other genes such as MDM2 and FRS2 also could provide proliferative advantages. The 12q-amplified neuroblastomas exhibit distinct clinical features and may benefit from targeted therapy using a small molecule CDK4/CDK6 inhibitor such as LEE011 (Novartis).

Citation Format: Susanne M. Fransson, Hanna Kryh, Niloufar Javanmardi, Inge M. Ambros, Ana P. Berbegall, Ingrid Ora, Rosa Noguera, Jurate Asmundsson, Bengt Sandstedt, Peter F. Ambros, Per Kogner, Tommy Martinsson. Amplification of CDK4 and MDM2 is associated with atypical clinical features in high risk neuroblastoma patients. [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 2435.
Abstract 2451: Cancer stem cell survival postradiation in medulloblastomas requires YAP, YB1, and IGF2
Authors: Dey Abhinav , Maier Caroline , Malhotra Anshu , Kenney Anna
Published: July 2016

Sonic hedgehog (Shh)-mediated medulloblastoma growth requires IGF2 (Insulin-like Growth Factor 2) and we recently showed that Yes Associated Protein (YAP1) induces IGF2 expression by Y-box protein 1(YB1) in Shh-stimulated cerebellar granule neural precursors (CGNPs), proposed cells-of-origin for the Shh molecular subclass of medulloblastoma, and mouse Shh-medulloblastoma cells. We found elevated levels of YAP1, YB1 and IGF2 in tumor cells occupying the peri-vascular niche, a microenvironmental niche proposed to house so-called tumor re-populating cells that survive radiation and contribute to medulloblastoma recurrence, which is fatal. We have developed an ex vivo approach using organotypic brain tumor slice cultures to better understand how YAP1, YB1, and IGF2 regulate peri-vascular niche cell survival post-radiation. We observed that the perivascular niche cell population expressing stem cell markers increases markedly following exposure to radiation. Additionally, on targeting any component of the YAP1-YB1-IGF2 axis we observed increased level of cell death within the niche and compromised cancer stem cell niche expansion post-radiation. These findings strongly indicate that therapeutic approaches intended to impair the function of this pathway could be used to reduce the use of cranio-spinal radiation of medulloblastoma patients, which causes life-long side effects that drastically impair quality of life.

Citation Format: Abhinav Dey, Caroline Maier, Anshu Malhotra, Anna Kenney. Cancer stem cell survival postradiation in medulloblastomas requires YAP, YB1, and IGF2. [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 2451.
Abstract 2456: Synergy between loss of NF1 and overexpression of MYCN in neuroblastoma is mediated by the GAP-related domain
Authors: He Shuning , Mansour Marc R. , Zimmerman Mark W. , Ki Dong Hyuk , Layden Hillary M. , Akahane Koshi , de Groh Eric D. , Perez-Atayde Antonio R. , Zhu Shizhen , Epstein Jonathan A. , Look A Thomas
Published: July 2016

Earlier reports indicated that the role of Nf1 tumor suppressor gene in limiting sympathoadrenal cell growth during embryologic development is independent of its ability to down-modulate RAS-MAPK signaling. This finding raised the question of whether neuroblastoma pathogenesis was also accelerated by loss of a similar non-canonical function of NF1. To elucidate how loss of the NF1 tumor suppressor gene contributes to the development of high-risk neuroblastoma, we relied on a transgenic zebrafish model that overexpresses MYCN and harbors loss-of-function nf1 mutations. We show here that loss of nf1 leads to aberrant activation of RAS signaling in MYCN-induced neuroblastoma, promoting both increased tumor cell survival and rapid tumor cell proliferation. We demonstrate further that the GTPase-activating protein (GAP) activity of the (GAP)-related domain (GRD) is sufficient to suppress accelerated initiation of neuroblastoma in nf1-deficient zebrafish, even though this transgene is unable to restrict abnormal sympathoadrenal cell growth during embryologic development. Hence NF1 exhibits different activities in vivo in the normal development and tumorigenesis of the peripheral sympathetic nervous system. Our findings establish nf1-deficient zebrafish that overexpress MYCN as an ideal animal model system for investigating new strategies to improve treatment of very high risk neuroblastomas with aberrant RAS-MAPK activation. We are currently performing high-throughput in vivo drug analysis using these zebrafish with primary tumors.

Citation Format: Shuning He, Marc R. Mansour, Mark W. Zimmerman, Dong Hyuk Ki, Hillary M. Layden, Koshi Akahane, Eric D. de Groh, Antonio R. Perez-Atayde, Shizhen Zhu, Jonathan A. Epstein, A Thomas Look. Synergy between loss of NF1 and overexpression of MYCN in neuroblastoma is mediated by the GAP-related domain. [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 2456.
Abstract 2460: Characterizing PAX3-FOXO1 dependent cell death in alveolar rhabdomyosarcoma reveals novel strategies for combination therapy
Authors: Wachtel Marco , Ommer Johannes , Schäfer Beat
Published: July 2016

Rhabdomyosarcoma is the most common soft tissue sarcoma in children. The aggressive alveolar subtype (aRMS) is characterized by chromosomal translocations, most often by t(2;13) resulting in the expression of the oncogenic fusion protein PAX3-FOXO1. Expression of this chimaeric transcription factor is critical for tumorigenesis and cell survival. However, the exact mechanism of cell death after loss of PAX3-FOXO1 activity has not been determined so far.

Our aim was thus to characterize the cell death pathways activated upon silencing of PAX3-FOXO1. In addition, we aimed at finding drugs further sensitizing aRMS cells to this mode of cell death.

We used combined shRNA and CRISPR approaches, as well as a small molecule screen to demonstrate that after shRNA-mediated silencing of PAX3-FOXO1 expression, aRMS cells undergo intrinsic apoptosis in a NOXA-dependent manner. In accordance, we can show that the BH3-mimetic ABT-263 sensitizes aRMS cells to PAX3-FOXO1 silencing via this cell death pathway. Interestingly, ABT-199 was less effective, suggesting that Bcl-xl plays a major anti-apoptotic role in aRMS. Furthermore, induction of cell death is dependent on PI3K activity and antagonized by GSK3, suggesting that inhibition of PI3K in this sarcoma might have an anti-apoptotic impact. In contrast, combination of ABT-263 or GSK-3 inhibitors with small molecule drugs affecting PAX3-FOXO1 activity such as PLK1 and aurora kinase inhibitors can cooperate to enhance cell death in aRMS cells. These studies demonstrate the importance of elucidating biological mechanisms to guide development of rational drug combinations.

Citation Format: Marco Wachtel, Johannes Ommer, Beat Schäfer. Characterizing PAX3-FOXO1 dependent cell death in alveolar rhabdomyosarcoma reveals novel strategies for combination therapy. [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 2460.
Abstract 2465: Spleen tyrosine kinase (SYK) in neuroblastoma tumorigenesis
Authors: Tuemmler Conny , Dumitriu Gianina , Cserna Julia , Moens Ugo L. , Kogner Per , Johnsen John Inge , Sveinbjørnsson Baldur
Published: July 2016

Tyrosine kinases play an important role in tumorigenesis by contributing to the regulation of cell proliferation and survival, and are therefore among the most attractive drug targets. SYK is a non-receptor tyrosine kinase highly expressed in cells of hematopoietic origin where it contributes to immune receptor signaling. The role of SYK in cancer is best understood in hematological malignancies where it promotes survival, and SYK inhibition has been shown to be a promising therapeutic approach. For solid tumors the role of SYK remains double-edged, as it has been found to promote but also suppress tumor development dependent on the cancer type. Besides its role in survival signaling SYK has also been found to contribute to drug resistance in several malignancies.

Neuroblastoma (NB) is a malignancy of early childhood. Although there have been great advances in the treatment of NB during the past 30 years, the prognosis for high- risk patients remains poor.

In this study we have evaluated SYK as a potential drug target in NB.

Examination of gene expression datasets indicated a correlation between high expression of SYK and poor prognosis in NB. Screening of NB cell lines showed that SYK mRNA was present in the majority of NB cells lines whereas the SYK protein was detected less frequently. However, SYK was detectable in NB tumor tissue using immunohistochemistry.

The efficacy of different, commercially available, SYK inhibitors was evaluated by comparing the neuroblastoma cell lines SH-SY5Y (high SYK expression) and SK-N-BE(2) (low SYK expression) using a cell viability assay. While one inhibitor displayed a markedly stronger effect on the viability of SH-SY5Y cells compared to SK-N-BE(2) the effect of the other inhibitors appears to be less specific. To confirm that SYK inhibition leads to decreased cell proliferation SYK specific siRNA was used. After 48h incubation the cell viability of SH-SY5Y cells was decreased by approximately 25%.

Our findings indicate that SYK may be a possible drug target in neuroblastoma. To further evaluate its potential, combination studies using SYK inhibitors and cytostatic drugs will be performed.

Citation Format: Conny Tuemmler, Gianina Dumitriu, Julia Cserna, Ugo L. Moens, Per Kogner, John Inge Johnsen, Baldur Sveinbjørnsson. Spleen tyrosine kinase (SYK) in neuroblastoma tumorigenesis. [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 2465.
Abstract 2466: Development of a novel gene targeting and clone screening platform to engineer common pediatric glioma mutations into model cell lines
Authors: Fons Nathan R. , Surovtseva Yulia , Breuer Gregory A. , Sundaram Ranjini K. , Bindra Ranjit S.
Published: July 2016

The utility of CRISPR/Cas9 gene targeting tools to alter endogenous genomic loci has revolutionized biomedical research across numerous disease focus areas, with especially large gains made in the development of disease models. However, the introduction of specific mutations using CRISPR/Cas9 can be associated with low yields of single cell clones harboring the desired homozygous or heterozygous mutations. Conventional clone screening methods can be a labor intensive and expensive process and thus, better approaches are needed to identify cells with the desired genotype(s). In parallel, there is a dearth of human tumor cell model systems for pediatric cancers, especially for Diffuse Intrinsic Pontine Glioma (DIPG). To address these needs, we recently designed and validated a platform which utilizes a novel, inducible Cas9 expression system coupled with a high-resolution DNA melt (HRM) analysis protocol to rapidly identify mutant clones. We applied our platform to model several common DIPG mutations, including truncating mutations within exon 6 of the gene, protein phosphatase 1D (PPM1D). Using our HRM analysis protocol, clones containing activating mutations in PPM1D were detectable and grouped distinctly from wild-type and non-functional clones; as validated through conventional Sanger sequencing and Ion Torrent Next Generation Sequencing. Importantly, multiple PPM1D mutant clones were generated in an expedited manner (< 4 weeks), using our unique HRM process-flow. We subsequently validated these clones in a collection of secondary assays, and we confirmed that the mutant proteins were functionally active. Several unique findings were identified in these studies, including substantial cell cycle-associated defects and basal activation of key proteins in the DNA damage response network. To our knowledge, we have created the first set of astrocyte cell lines harboring engineered PPM1D mutations at the endogenous gene locus. We are now testing these cell lines in high-throughput synthetic lethal screens to identify novel inhibitors of PPM1D-mutant DIPG tumors. These findings highlight the utility and power of our unique approach to rapidly create pertinent mutant cell lines for use in mutation-targeted biological assays. Our platform likely will become an invaluable tool for the development of better pediatric glioma cell line models in the future.

Citation Format: Nathan R. Fons, Yulia Surovtseva, Gregory A. Breuer, Ranjini K. Sundaram, Ranjit S. Bindra. Development of a novel gene targeting and clone screening platform to engineer common pediatric glioma mutations into model cell lines. [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 2466.
Abstract 2485: Heterozygous deletion of RUNX3 promotes vasculogenesis by enhancing endothelial progenitor cells function
Authors: Oh Su Young , Lee Se-Hyung , Lee Sun Hee , Lee You Mie
Published: July 2016

Endothelial progenitor cells (EPCs) are bone marrow (BM) -derived stem cells to be committed and differentiated into mature endothelial cells. Runt-related transcription factor 3 (RUNX3) is a tumor suppressor involved in TGF-β signaling pathway and participate in cell cycle arrest and anti-angiogenesis. RUNX3 is frequently deleted or transcriptionally silenced in a variety of cancers by epigenetic mechanisms. The aim of study was to determine the role of RUNX3 in EPC function and vasculogenesis. Here we found that RUNX3 homozygous knockout (Rx3-/-) embryos had vascular defects in embryonic day (ED) 10 and developmental abnormalities in ED 16.5 organs. The number of EPC colonies and circulating EPCs (CD34+/VEGFR2+ cells), migration and tube formation capacity of EPCs derived from RUNX3 heterozygous (Rx3+/-) mice were increased in compared to those wild type (WT) mice, suggesting that deletion of RUNX3 can enhance vasculogenesis. Expression level of VEGF, VEGFR2, SDF-1, CXCR4 and endothelial nitric oxide synthase (eNOS) mRNA was significantly up-regulated in Rx3+/- EPCs. Protein expression of VEGF, p-VEGFR2, p-Akt, p-ERK, p-SAPK/JNK and p-eNOS was also augmented in Rx3+/- EPCs. Finally, we found that recovery of blood flow was highly stimulated in Rx3+/- mice using hindlimb ischemia mouse models. Taken together, our study revealed that RUNX3 might inhibit vasculogenic processes in physiological ischemia by inhibition of EPC functions.

Citation Format: Su Young Oh, Se-Hyung Lee, Sun Hee Lee, You Mie Lee. Heterozygous deletion of RUNX3 promotes vasculogenesis by enhancing endothelial progenitor cells function. [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 2485.
Abstract 2488: Stat3 inhibitor (BBI-608) with radiation therapy is promising in malignant pleural mesothelioma
Authors: Matsumoto Seiji , Doi Hiroshi , Nakamichi Tohoru , Kuroda Ayumi , Hashimoto Masaki , Takuwa Teruhisa , Kondo Nobuyuki , Hasegawa Seiki
Published: July 2016

Background:

The prognosis of Malignant pleural mesothelioma (MPM) is very poor, the new drug for MPM is need. IL-6 in serum with MPM patients is high, IL-6/Stat3 pathway is activated. We investigated that Stat3 is the potential target for the treatment of MPM.

Methods:

Cell viability was assayed with Cell Counting Kit-8 (CCK-8: WST-8 Dojindo). MPM cells (NCI-H28, NCI-H226, NCI-H2052, NCI-H2452, MSTO-211H) were seeded into 96-wellplates. After the treatment with Stat3 inhibitor (BBI-608). Cell Counting Kit-8 solution was added to each well of the plate and measured the absorbance using a microplate reader. The levels of phosphorylated Stat3 (p-Stat3) were measured in cell lysates using an InstantOne ELISA assays (eBioscience). The translocated p-Stat3, c-Myc were analyzed by Confocal immunofluorescent. In vivo study, H226 cells were injected into the subcutaneous over the flank region of nude mice. Mice were randomly assigned into four groups (5 mice each group) 1) vehicle control 2) treated with BBI-608 3) Radiation 4) BBI608/RT. Tumor is measured twice per week.

Results:

BBI-608 inhibited MPM cell lines (NCI-H28, NCI-H226, NCI-H2052, NCI-H2452, MSTO-211H) viability in a dose-dependent manner. The level of p-Stat3 was decreased 90% by BBI-608 10μM treatment in H226. Untreated H226, p-stat3 was observed in cytoplasma and localized in the nucleus. As compared with untreated cells, p-stat3, c-Myc were decrease in cytoplasma and was not localized in the nucleus with BBI-608 treated cells. BBI-608 suppressed tumor growth (p<0.05) and completely suppressed tumor growth with radiation in mouse model.

Conclusion:

In this study, we have shown that BBI-608 inhibited the proliferation of all MPM cell (epithelial, biphasic, sarcomatoid) lines and inhibited Stat3 phosphorylation and blocked the translocation to the nucleus. We also demonstrated that BBBI-608 completely suppressed tumor growth with radiation in mouse model. Furthermore, Stat3 inhibitor with Radiation Therapy (START) is promising in MPM therapy.

Citation Format: Seiji Matsumoto, Hiroshi Doi, Tohoru Nakamichi, Ayumi Kuroda, Masaki Hashimoto, Teruhisa Takuwa, Nobuyuki Kondo, Seiki Hasegawa. Stat3 inhibitor (BBI-608) with radiation therapy is promising in malignant pleural mesothelioma. [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 2488.
Abstract 2499: Sox2 enhances breast tumor angiogenesis by promoting the transition of cancer cell to CD31+ and LYVE-1+ cells
Authors: Wang Rongrong , Li Xuefei , Xu Yingxi , Sun Tongchao , Su Weijun , Yu Chenhua , Dong Xiaoli , Xiang Rong , Li Na
Published: July 2016

Angiogenesis plays an important role during the development of tumor. Sox2 is one of the transcription factors involved in tumorigenesis and metastasis. However, its function during the process of tumor angiogenesis and lymphogenesis is still poorly understood. Here, we demonstrated that down-regulation of Sox2 significantly reduced the tumor angiogenesis and lymphogenesis as reflected by attenuated immunofluorescence staining of CD31 and LYVE-1 and improved prognosis in xenograft models of breast tumor. Furthermore, Sox2 also promoted the recruitment of bone marrow derived endothelial progenitor cells (MDEPC) to tumor microenvironment. Most importantly, we demonstrated that some of CD31+ and LYVE-1+ cells were derived from Sox2+ cancer cells and silencing of Sox2 could inhibit this transition. We revealed that Sox2 promoted the expression of LYVE-1, VEGFs and enhanced the phosphorylation of VEGFR2 in breast cancer. ChIP-seq and dual-luciferase assay further revealed the binding of Sox2 protein on proximate promoter region of VEGFB and LYVE1 to regulate their transcriptional activities, disclosing the underlying molecular mechanism on the regulatory effects of Sox2 on angiogenesis and lymphogenesis. Our study indicated that Sox2 improved tumor angiogenesis and lymphogenesis through regulating the related genes and unveiled a novel function of Sox2 in tumor progression.

Citation Format: Rongrong Wang, Xuefei Li, Yingxi Xu, Tongchao Sun, Weijun Su, Chenhua Yu, Xiaoli Dong, Rong Xiang, Na Li. Sox2 enhances breast tumor angiogenesis by promoting the transition of cancer cell to CD31+ and LYVE-1+ cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2499.
Abstract 25: SCAP links glucose to lipids for tumor growth
Authors: Cheng Chunming , Geng Feng , Guo Jeffrey Yunhua , Wu Xiaoning , Cheng Xiang , Chakravarti Arnab , Guo Deliang
Published: July 2016

Increased glucose consumption and elevated lipogenesis are co-occurred in malignancies. But the molecular mechanism of the link between glucose and lipid metabolism remains elusive. Our previous studies have uncovered that sterol regulatory element-binding protein (SREBP-1), an endoplasmic reticulum-bound transcription factor with a central role in lipid metabolism, is highly upregulated in glioblastoma (GBM), the most deadly brain tumor. In the current study, we found that SREBP-cleavage activating protein (SCAP), a key player regulating SREBP trafficking from ER to the Golgi and subsequent SREBP activation, links oncogenic signaling and glucose consumption to lipid metabolism. Our data showed that glucose is a critical activator for SREBP function via N-glycosylation of SCAP. N-glycosylation is a key signal to promote SCAP/SREBP complex move from ER to the Golgi and SREBP activation. Moreover, we found that EGFR/PI3K/Akt signaling activates SREBP-1 via upregulation of SCAP N-glycosylation and its protein levels through enhancing glucose uptake. Genetically silencing SCAP or mutation of the N-glycosylation sites on SCAP downregulates SREBP-1 activity and impairs GBM tumor growth. Taken together, our study revealed that glucose is a critical activator for SREBP-1 function and lipid metabolism, and SCAP integrates oncogenic signaling and fuel availability to SREBP-1-mediated lipogenesis for tumor growth. Our study also demonstrated that targeting SCAP N-glycosylation represents a promising therapeutic strategy to treat malignancies.

Citation Format: Chunming Cheng, Feng Geng, Jeffrey Yunhua Guo, Xiaoning Wu, Xiang Cheng, Arnab Chakravarti, Deliang Guo. SCAP links glucose to lipids for tumor growth. [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 25.
Abstract 2500: Grape compounds suppress colon cancer stem cells in vitro and in a rodent model of colon carcinogenesis
Authors: Reddivari Lavanya , Charepalli Venkata , Vadde Ramakrishna , Radhakrishnan Sridhar , Lambert Joshua D. , Elias Ryan J. , Vanamala. Jairam KP
Published: July 2016

We have previously shown that the grape bioactive compound resveratrol (RSV) potentiates grape seed extract (GSE)-induced colon cancer cell apoptosis at physiologically relevant concentrations. However, RSV-GSE combination efficacy against colon cancer stem cells (CSCs), which play a key role in chemotherapy and radiation resistance, is not known. Hence, we tested the anti-cancer efficacy of the RSV-GSE in an azoxymethane-induced mouse model of colon carcinogenesis. RSV-GSE suppressed tumor incidence similar to sulindac, without any gastrointestinal toxicity. Additionally, RSV-GSE treatment reduced the number of crypts containing cells with nuclear β-catenin (an indicator of colon CSCs) via induction of apoptosis. In in vitro mechanistic studies, RSV-GSE suppressed proliferation and sphere formation properties of colon CSCs (positive for CD44, CD133 and ALDH1b1), suppressed pGSK3β and nuclear translocation of β-catenin, a critical regulator of CSC proliferation, similar to sulindac. RSV-GSE, but not sulindac, suppressed nuclear levels of downstream proteins of Wnt/β-catenin pathway, c-Myc and cyclin D1. RSV-GSE also induced mitochondrial-mediated apoptosis in colon CSCs characterized by elevated p53, Bax/Bcl-2 ratio and cleaved PARP. Furthermore, shRNA-mediated knockdown of p53, a tumor suppressor gene that is mutated in advanced stages of colon cancer, in the colon CSCs did not alter efficacy of RSV-GSE. The suppression of Wnt/β-catenin signaling and elevated mitochondrial-mediated apoptosis in colon CSCs support potential clinical testing/application of grape bioactives for colon cancer prevention and/or therapy.

Citation Format: Lavanya Reddivari, Venkata Charepalli, Ramakrishna Vadde, Sridhar Radhakrishnan, Joshua D. Lambert, Ryan J. Elias, Jairam KP Vanamala. Grape compounds suppress colon cancer stem cells in vitro and in a rodent model of colon carcinogenesis. [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 2500.
Abstract 2501: Combination therapy with mebendazole, trametinib and metformin eliminates recalcitrant NRASQ61K melanoma cells
Authors: AbdusSamad Maryam , Gaur Anirudh , Simbulan-Rosenthal Cynthia M. , McCarron Edward C. , Rosenthal Dean S.
Published: July 2016

Melanoma is an aggressive and lethal form of cancer with increasing incidence worldwide. In recent years, the FDA approved several promising treatments for malignant melanoma. However, treatment often fails due to the survival and regrowth of drug-resistant cells. Studies by our lab and others suggest that Melanoma Initiating Cells (MIC), a CD133+ population that forms xenograft tumors, may represent an intermediate stage in the acquisition of stable drug resistance. This study aims to determine the effect of CD133-expression and drug resistance in metastatic melanoma cells harboring difficult-to-treat mutation signatures by focusing on the two main downstream cascades of MAPK and PI3K/AKT/mTOR. Our results show that triple combination therapy using mebendazole (repurposed anthelmintic BRAF inhibitor), trametinib (MEK inhibitor) and metformin (repurposed anti-diabetic mTOR inhibitor) synergistically reduces cell viability of NRASQ61K melanoma cells when compared with mebendazole, trametinib or metformin alone. Further, the surviving fraction from each monotherapy is highly enriched for CD133+ cells. Thus, the multi-kinase inhibitor trametinib, along with repurposed anthelmintic and anti-diabetic drugs may inhibit MAPK and PI3K/AKT/mTOR pathways, which could potentially be an effective therapy for the resistant subpopulation of melanoma initiating cells.

Citation Format: Maryam AbdusSamad, Anirudh Gaur, Cynthia M. Simbulan-Rosenthal, Edward C. McCarron, Dean S. Rosenthal. Combination therapy with mebendazole, trametinib and metformin eliminates recalcitrant NRASQ61K melanoma cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2501.
Abstract 2502: Bioengineered-induced cancer-like stem cells: characterization and applications
Authors: Cho Sungpil , Park Hongsuk , Jarboe Elke A. , Peterson C Matthew , Bae You Han , Janat-Amsbury Margit Matthew
Published: July 2016

Cancer stem cells (CSCs) are a small subset of tumor cells capable of self-renewal, tumor initiation, and growth. Isolation, expansion, and differentiation of CSCs in the proper stem niches remain a topic of great debate. To address some of the challenges, we developed bioengineered-induced cancer like stem cells (iCLSCs) through in vitro oncogenic reprograming of C57BL/6 mouse embryonic stem cells (mESCs). We constructed retroviral plasmids through integration of well-defined oncogenic elements such as SV40LTg and HrasV12 into a mouse stem virus long terminal repeat (MSCV-LTR) plasmid fused with either a GFP or RFP gene. We successfully acquired these bioengineered-iCLSCs through retroviral introduction alongside with FACS sorting of positively infected mESCs. We then cultured the bioengineered-iCLSCs on mouse embryonic fibroblast (mEF) feeder cells. Bioengineered-iCLSCs expressed the introduced oncogenic genes and showed enhanced proliferation similar to cancer cells. In addition, we ascertained the unhampered stem properties of the bioengineered-iCLSCs by examining the maintenance of alkaline phosphatase activity as well as the expression of mouse stem cell marker SSEA-1. Furthermore, the orthotopic inoculation of these bioengineered-iCLSCs into either the mammalian fat pad or the ovarian bursa of female C57BL/6 mice lead to the formation of teratomas in supportive niches. Interestingly, the teratomas formed in the ovarian bursa exhibited malignant and immature features, whereas the iCLSCs introduced to the mammary fat pad were not able to generate teratomas with similar features. These observations call for additional research to identify the dependency on a supportive niche and their role in metastasis based on a tumor-favorable microenvironment. Our study helped to establish the use of in vitro oncogenic reprograming to generate bioengineered-iCLSCs and suggests the potential application for the initiation of organ-specific, malignant tumor formation in an orthotopic small animal cancer model.

Citation Format: Sungpil Cho, Hongsuk Park, Elke A. Jarboe, C Matthew Peterson, You Han Bae, Margit Matthew Janat-Amsbury. Bioengineered-induced cancer-like stem cells: characterization and applications. [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 2502.
Abstract 2503: NQO1's role in maintaining the cancer stem cell phenotype in NSCLC
Authors: Madajewski Brian , Bey Erik A.
Published: July 2016

NAD(P)H:quinone oxidoreductase 1 (NQO1) is a phase II detoxifying enzyme responsible for quinone reduction, where it scavenges quinone induced reactive oxygen species. In recent studies, NQO1 has been investigated as a possible drug target due to its overexpression in a number of solid tumors. To this end, treatment with the quinone-analog drug, β-lapachone (ARQ-761), has progressed to Phase II clinical trials. In an effort to better understand the necessity of NQO1 in the overall lifecycle of cancer, our earlier work demonstrated that NQO1 played a vital role in a number of tumorigenic processes including anoikis resistance, and most interestingly alteration of aldehyde dehydrogenase (ALDH) activity. Given that ALDH is a widely reported cancer stem cell marker, and loss of NQO1 leads to a decrease in ALDH activity, we have begun to investigate the effect of NQO1 expression on the non-small cell lung cancer (NSCLC) stem cell population. Here, we present data that demonstrates NQO1 is vital to tumorsphere formation as demonstrated by decreased spheroid formation following NQO1 knocked down with shRNA. We also show that NQO1 appears to be necessary for cancer stem cell renewal as illustrated by decreased serial tumorsphere formation. In addition to these aforementioned data, in extreme limited dilution assays we demonstrate a reduced cancer stem cell frequency in NQO1 knockdown cells as compared to controls. Interestingly, in NQO1 knockdown populations, those cells that do form spheres show a remarkable re-expression of NQO1 as well as a rescue of ALDH activity, further supporting NQO1's role in tumorsphere formation and stem cell maintenance. Future work on this project will involve use of the CRISPR-Cas9 system to generate NQO1 knockout cell lines, drug resistance studies, cancer stem cell marker validation, and in vivo limited dilution assays to definitively demonstrate NQO1's necessity in maintaining the NSCLC stem cell population.

Citation Format: Brian Madajewski, Erik A. Bey. NQO1's role in maintaining the cancer stem cell phenotype in NSCLC. [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 2503.
Abstract 2511: Association of ALDH1A1 with cisplatin resistance via modulation of NEK-2 in ovarian cancer stem-like cells
Authors: Uddin Md. Hafiz , Song Yong Sang
Published: July 2016

Cisplatin (CDDP) resistance is the foremost obstacle in the treatment of ovarian cancer patients. According to the cancer stem cell hypothesis, the recurrence and chemoresistance are presumed to be linked to cancer stem/progenitor cells. Here, we investigated the cancer stem cell like phenotypes and mechanism of chemoresistance in CDDP resistant ovarian cancer cells.

A well-established CDDP sensitive ovarian cancer cell line A2780 and its resistant clone A2780 Cp were used and we also developed a supra resistant clone (SKOV3 Cp) from a naturally CDDP resistant cell line SKOV3 by six shocks treatment. Sensitiveness to CDDP was evaluated by MTT assay and self renewal efficiency by sphere formation, noble agar and clonogenic assays. A few cancer hallmarks and stem-cell markers were characterized using flow cytometry. Molecular analysis was performed using quantitative PCR and western blotting. For the silencing of selected genes small interfering RNA (siRNA) based approaches was utilized.

Both CDDP resistant A2780 Cp and supra resistant SKOV3 Cp cell lines showed significantly (P < 0.05) higher self-renewal ability in sphere formation, noble agar and clonogenic assays than their parental counterparts. They also showed significant resistance to apoptosis and sub-G1 arrest upon CDDP treatment. Stem cell marker ALDH1 positivity rates were higher both in A2780 Cp and SKOV3 Cp cell lines, quantified by ALDEFLUORTM assay kit using flow cytometry. Hoechst 33342 dye effluxing side populations were increased about five folds in A2780 Cp cells and two folds in SKOV3 Cp cells compared to A2780 and SKOV3 cells respectively. Among major stemness related genes (OCT4, SOX2, NANOG, NESTIN, BMI1, KLF4 and ALDH1A1) quantitative PCR analysis showed significantly higher expression of ALDH1A1 and KLF4 genes in both resistant and supra resistant cells than their parental clones. Of two genes, silencing ALDH1A1 in A2780 and A2780 Cp cells using siRNA greatly reduced the stem cell population as detected by ALDEFLUORTM assay and sensitized cells to CDDP. Moreover, silencing of ALDH1A1 reduce the level of its downstream target NEK-2. We also observed the downregulation of ABC transporters (ABCB1, ABCG2 and ABCC1) by silencing either ALDH1A1 or NEK-2 gene.

Taken together, present study showed for the first time that stemness gene ALDH1A1 can be involved in CDDP resistance via the upregulation of NEK-2 in ovarian or solid cancer.

Citation Format: Md. Hafiz Uddin, Yong Sang Song. Association of ALDH1A1 with cisplatin resistance via modulation of NEK-2 in ovarian cancer stem-like cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2511.
Abstract 2515: Pharmacological WNT-inhibition acts synergistically with chemo- and radiotherapy by overcoming treatment-resistance in glioma stem cells
Authors: Suwala Abigail K. , Kahlert Ulf D. , Maciaczyk Jaroslaw
Published: July 2016

Objective: Glioma Stem Cells (GSCs) are considered to be responsible for Glioblastoma's (GBM’s) dismal prognosis. WNT signaling is one of the major players in GSCs that promotes their radio- and chemoresistance. In this study we tested whether LGK974, an inhibitor of both canonical and non-canonical WNT signaling, might sensitize GBM cells to chemotherapeutics and irradiation and determined possible mechanisms causing treatment-resistance in GBM.

Methods: At first we analyzed MGMT promoter methylation status of several GBM cell lines in order to define cells resistant to the standard chemotherapeutic drug Temozolomide (TMZ). For further investigations we chose two cell lines resistant or sensitive to TMZ, respectively. IC50 concentrations of TMZ and LGK974 as well as IC50 dose of γ-irradiation were tested via Titer Blue cell viability assay. Effectiveness of different concentration of each single therapy and its combination with LGK974 was determined to create a drug-response-curve due to the computerized simulation of synergism and antagonism in drug combination studies (Chou, 2006). A reporter assay for canonical WNT/β-catenin signaling was used to quantify the effect of TMZ and irradiation alone and in combination with LGK974 on the canonical and non-canonical WNT pathway. Western Blot and qPCR were applied to analyze the effects of combinatory therapy on protein and gene expression concerning WNT pathway activation.

Results: We observed a significant synergy of combined irradiation/TMZ with LGK974 in all cell lines. There was no difference in effectiveness between cell lines resistant or sensitive to TMZ. Interestingly, following TMZ treatment or irradiation cells showed slightly decreased canonical WNT pathway activity. Furthermore, non-canonical target genes and receptors were upregulated under chemo- and radiotherapy, suggesting a reciprocal activation of the non-canonical branch of WNT signaling pathway. This phenomenon might possibly be responsible for the development of resistance against standard chemo- and radiotherapy.

Conclusions: We propose that GSCs undergo a shift from canonical to non-canonical WNT signaling to become treatment-resistant under standard chemo- and radiotherapy. Treatment with LGK974 results in therapeutic synergy at least in part via suppressing the therapy-induced activation of non-canonical WNT signaling. Our results confirm the potential benefit of combining TMZ or irradiation with the WNT inhibitor LGK974 to overcome therapy-resistance in GBM.

Citation Format: Abigail K. Suwala, Ulf D. Kahlert, Jaroslaw Maciaczyk. Pharmacological WNT-inhibition acts synergistically with chemo- and radiotherapy by overcoming treatment-resistance in glioma stem cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2515.