bluuebunny/crossref_metadata_2025_split huggingface dataset:
Abstract 600: Melanoma and ovarian cancer cells tested for drug sensitivity using anchorage-independent growth
Authors: Rotem Asaf , Izar Benjamin , Garraway Levi A. 
Published: July 2016

Abstract
This study aims to test patient-derived melanoma and ovarian cancer cells for their sensitivity to clinically available drugs and drug combinations. The experiments were performed by using the growth in low attachment (GILA) assay, which is equivalent to the classic soft-agar assay and allows screening of 3D ex vivo cultures, which resemble the tumor and its microenvironment.
To assay tumor cells from malignant lesions we isolated ovarian cancer cells from peritoneal fluid (ascites) derived from patients and melanoma cells from metastatic melanoma patients. The fresh samples were enriched for tumor cells and later cultured in an ex vivo manner on either high- or low-attachment surfaces. These cells were subjected to drug treatments and cellular viability was measured at the end of these experiments.
Our experiments demonstrate that the GILA assay is a highly sensitive method for pharmacological tests, as compared to the traditional 2D method (growth on high-attachment surfaces). By using GILA assay we found that melanoma cells are sensitive to drugs that inhibit driver oncogenes, such as BRAF or MEK. In addition, we found interesting targets in metastatic melanoma lesions that carry an unknown mutational background.
Drug sensitivity assays for ovarian cancer cells using the GILA approach showed that cancer cells from various patients with the same cancer type display differential sensitivity to drugs. Additionally, ovarian cancer cells were found to have a different drug-sensitivity threshold when grown in spheres.
Our studies present an approach that utilizes the unique ability of transformed cells to grow in anchorage-independent conditions and enables ex vivo drug sensitivity testing. This phenotypic approach is complementary to a genetic approach that uses DNA sequencing to identify putative oncogenes that confer sensitivity to drugs designed to specifically inhibit the identified oncoprotein.
Citation Format: Asaf Rotem, Benjamin Izar, Levi A. Garraway. Melanoma and ovarian cancer cells tested for drug sensitivity using anchorage-independent 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 600.
Abstract 602: BEZ235 efficacy in 3D-cancer cell line screening
Authors: Irigoyen Macarena , Castillo Gonzalo 
Published: July 2016

Abstract
In vitro efficacy assessment of drugs in panels of tumor-derived cell lines has proven to be a powerful tool for predictive biomarker discovery. However, in the last few years it has become evident that particularly for tumor cell lines derived from solid tumors, the results do not always correlate with what is seen in xenografts as well as clinically. One of the reason is that traditionally these type of studies are done in monolayers (2D), which lack the complexity normally seen in vivo. 3D cultures offer the ability to carry out the same type of studies with the added benefit of incorporating 3-dimensional cellular interactions and physiological gradients, normally seen in a tumor microenvironment. In this study, we evaluated the efficacy of BEZ235, an imidazoquinoline, targeting the phosphatidylinositol 3 kinase (PI3K) and the mammalian target of rapamycin (mTOR). BEZ235 was evaluated in a panel of 60 cell lines grown as spheroids, and this information was used to investigate genes associated with sensitivity. Cell lines grown as spheroids proved to be highly sensitive to the drug, where 45 of 60 cell lines showed an IC50 lower than 1 μM. Biomarker association analysis identified two mutated genes associated to BEZ235 sensitivity. The first biomarker was the phosphatase and tensin homolog (PTEN) a tumor suppressor gene, that when deregulated plays an important role in the development of several cancers. The second biomarker was AXIN1, a negative regulator of the WNT signaling pathway, and its deregulation has been associated with hepatocellular carcinoma. Since both PTEN and AXIN1 are thought to play a role in tissue invasion and metastasis, we studied the ability of BEZ235 to inhibit cellular invasion, and the results are discussed.
Citation Format: Macarena Irigoyen, Gonzalo Castillo. BEZ235 efficacy in 3D-cancer cell line screening. [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 602.
Abstract 61: A genome-scale CRISPR-Cas9 screening method for protein stability reveals novel regulators of Cdc25A
Authors: Wu Yuanzhong , Kang Tiebang 
Published: July 2016

Abstract
The regulation of stability is particularly crucial for unstable proteins in cells. However, a convenient and unbiased method of identifying regulators of protein stability remains to be generated. Recently, genome-scale CRISPR-Cas9 library has been established as a genetic tool to mediate loss of function screening. Here, we developed a method named Protein Stability Regulators Screening Assay (Pro-SRSA) by combining the whole genome CRISPR-Cas9 library with a dual-fluorescence-based protein stability reporter and high-throughput sequencing to screen regulators of protein stability. Using Cdc25A as an example, Cul4B-DDB1-DCAF8 was determined to be a new E3 ligase for Cdc25A. Moreover, the acetylation of Cdc25A at lysine 150, which was mediated by p300/CBP and deacetylated by HDAC3, was revealed to prevent its ubiquitin-mediated degradation by proteasome. This is the first report that the acetylation as a novel posttranslational modification modulates the Cdc25A stability, and we believe that this efficient and unbiased screening method at the genome-scale will be widely used to globally identify regulators of protein stability.
Citation Format: Yuanzhong Wu, Tiebang Kang. A genome-scale CRISPR-Cas9 screening method for protein stability reveals novel regulators of Cdc25A. [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 61.
Abstract 62: Functional analysis of endometrial cancer mutants of the E3 ubiquitin ligase adaptor protein, SPOP
Authors: Lozy Fred , Urick Mary Ellen , Rudd Meghan , Maurer Deena , Bell Daphne W. 
Published: July 2016

Abstract
The purpose of this study is to functionally annotate endometrial cancer (EC) specific mutants of SPOP (Speckle-type POZ protein). SPOP is the substrate adaptor for the E3 ligase complex CUL3-SPOP-RBX1, which is responsible for mediating the ubiquitination, and subsequent proteasomal degradation of a number of protein substrates including the SRC3 (NCOA3/AIB1) oncoprotein. Our laboratory has previously shown that SPOP is frequently mutated in serous and clear cell ECs, two clinically aggressive subtypes of EC. Importantly, the vast majority of SPOP mutants localized within the MATH domain, which is responsible for substrate binding. We hypothesize that EC associated SPOP mutations encode loss-of-function mutants that abolish or attenuate substrate degradation.
To test this hypothesis, we initially focused our attention on five SPOP mutations we uncovered in ECs (E47K, S80R, P94A, M117I and R121Q). HEK293 cells were transiently co-transfected with a HA-tagged SRC3-expressing construct and a MYC-tagged construct expressing either vector alone, wildtype SPOP, or mutant forms of SPOP. Protein lysates from transfected cells were subjected to Western blotting to measure exogenous levels of HA-SRC3 and MYC-SPOP. Cells exogenously expressing wildtype SPOP or the E47K, P94A, M117I or R121Q mutants exhibited lower levels of HA-SRC3 than cells expressing the vector control. In contrast, cells expressing the S80R mutant had elevated levels of HA-SRC3 compared to cells expressing either wildtype SPOP or the vector control. Co-immumoprecipitation experiments are being performed to assess the substrate binding capacity of SPOP mutants.
Our findings indicate that the SPOP-S80R mutation, which is recurrent in EC, has an impaired ability to down-regulate SRC3 protein levels compared to wildtype SPOP.
Ongoing and future studies aim to elucidate the mechanism that underlies the effect of SPOP-S80R, and to functionally annotate additional SPOP mutations in EC.
Citation Format: Fred Lozy, Mary Ellen Urick, Meghan Rudd, Deena Maurer, Daphne W. Bell. Functional analysis of endometrial cancer mutants of the E3 ubiquitin ligase adaptor protein, SPOP. [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 62.
Abstract 634: Impact of tumor inoculation site on the development of cancer cachexia
Authors: Ando Takashi , Ishikawa Takeshi , Matsuyama Tastuzo , Yasuda Tomoyo , Doi Toshifumi , Okayama Tetsuya , Sakamoto Naoyuki , Kokura Satoshi , Naito Yuji , Itoh Yoshito 
Published: July 2016

Abstract
Background and Aim
The incidence of cancer cachexia varies greatly among tumor type. Although certain tumor types are more commonly associated with cachexia, even for the same tumor type, the degree to which patients exhibit cachexia varies. This is due to variations in tumor phenotype and host genotype that contributes to the development of cancer cachexia. The influence of the site of tumor growth on the development of cancer cachexia remains unclear. In this study, we evaluated if differences in tumor microenvironment would affect the development of cancer cachexia in a murine model.
Methods
The same number of colon26 cells (5×10⁁6 cells/mouse) was inoculated subcutaneously or intraperitoneally in 8-week-old male BALB/c mice. Body weight, food intake, and tumor size were monitored. On day 14 after the tumor inoculation, mice were anesthetized and blood collected, and the weight of peritesticular adipose tissue, gastrocnemius muscle and heart were measured. 23 plasma cytokines were determined by using the Bio-Plex array system and plasma Myostatin and Activin A levels were measured by ELISA. Expression of MuRF-1 and Antrogin-1, which are required for muscle atrophy, in gastrocnemius muscle or heart was assessed by RT-PCR.
Results
The body weight, adipose tissue and gastrocnemius muscle decreased in tumor-bearing mice. Interestingly, a reduction in heart weight was observed in the intraperitoneal tumor group but not in the subcutaneous group. The weight loss of the body, adipose tissue and gastrocnemius was more severe in the intraperitoneal group than in the subcutaneous group. Atrogin 1 and MuRF1 mRNA expressions in the gastrocnemius muscle increased significantly in both groups of tumor-bearing mice, however, in the myocardium, expression of these mRNAs increased in the intraperitoneal group but not in subcutaneous group. As for plasma cytokine levels, several cytokine levels including IL-6, TNF-α, and activing A of tumor-bearing mice were significantly higher than those of control mice. Eotaxin and G-CSF levels in the intraperitoneal tumor group were higher than in the subcutaneous group.
Conclusion
Based on these data, we believe that differences in microenvironment where tumor cells develop can affect the progression and phenotype of cancer cachexia through alterations in various circulating factors derived from the tumor microenvironment.
Citation Format: Takashi Ando, Takeshi Ishikawa, Tastuzo Matsuyama, Tomoyo Yasuda, Toshifumi Doi, Tetsuya Okayama, Naoyuki Sakamoto, Satoshi Kokura, Yuji Naito, Yoshito Itoh. Impact of tumor inoculation site on the development of cancer cachexia. [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 634.
Abstract 642: Hypoxia-regulated gene expression explains differences between cell line-derived xenografts and patient-derived xenografts
Authors: Bhadury Joydeep , Einarsdottir Berglind O. , Podraza Agnieszka , Bagge Roger Olofsson , Stierner Ulrika , Ny Lars , López Marcela Dávila , Nilsson Jonas A. 
Published: July 2016

Abstract
Cell line-derived xenografts (CDXs) are an integral part of drug efficacy testing during development of new pharmaceuticals against cancer but their accuracy in predicting clinical responses in patients have been debated. Patient-derived xenografts (PDXs) are thought to be more useful for predictive biomarker identification for targeted therapies, including metastatic melanoma, due to their similarities to human disease. Here, tumor biopsies from patients and ten widely-used melanoma cell lines were transplanted into immunocompromised mice to generate PDXs and CDXs, respectively. Gene expression profiles generated from the tumors of these PDXs and CDXs clustered into distinct groups, despite similar mutational signatures. Hypoxia-induced gene signatures and overexpression of the hypoxia-regulated miRNA hsa-miR-210 characterized CDXs. Inhibition of hsa-miR-210 with decoys had little phenotypic effect in vitro but resulted in reduced sensitivity to MEK1/2 inhibition in vivo, suggesting down-regulation of this miRNA could result in development of resistance to MEK inhibitors.
Citation Format: Joydeep Bhadury, Berglind O. Einarsdottir, Agnieszka Podraza, Roger Olofsson Bagge, Ulrika Stierner, Lars Ny, Marcela Dávila López, Jonas A. Nilsson. Hypoxia-regulated gene expression explains differences between cell line-derived xenografts and patient-derived xenografts. [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 642.
Abstract 643: Establishment and mutation analysis of a novel malignant peritoneal mesothelioma cell line, TU-MM-1
Authors: Sato Seiya , Oumi Nao , Itamochi Hiroaki , Oishi Tetsuro , Harada Tasuku , Sugiyama Toru 
Published: July 2016

Abstract
Malignant peritoneal mesothelioma (MPM) is a rare aggressive neoplasm of the peritoneum, and its molecular biological characteristics and mechanisms of development are poorly understood. We, therefore, established a new cell line of human MPM, TU-MM-1, to elucidate its biological properties and to develop novel therapeutic strategies. The cells showed polygonal morphology, grew in monolayers without contact inhibition and were arranged like a jigsaw puzzle. The chromosome numbers ranged from 41 to 44. A low rate of proliferation was observed and the doubling time was 67.9 h. Genomic DNA sequencing revealed that TU-MM-1 cells harbored missense mutations in APC, LATS2, BRCA1/2, and TP53, and mutation of a splice donor site in BAP1 and loss of CDKN2A gene. We observed the absence of BAP1 and p16INK4a proteins, underexpression of LATS2 protein, and overexpression of p53 protein in TU-MM-1 cells in western blot analysis. Heterotransplantation to nude mice produced tumors that had the characteristics of the original tumor. This cell line may be useful for studying biological properties and contribute to novel treatment strategies.
Citation Format: Seiya Sato, Nao Oumi, Hiroaki Itamochi, Tetsuro Oishi, Tasuku Harada, Toru Sugiyama. Establishment and mutation analysis of a novel malignant peritoneal mesothelioma cell line, TU-MM-1. [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 643.
Abstract 652: Establishment of a novel mouse model of intrahepatic cholangiocarcinoma by liver-specific Kras activation and Pten deletion
Authors: Terakado Yumi , Ikenoue Tsuneo , Yamaguchi Kiyoshi , Furukawa Yoichi 
Published: July 2016

Abstract
The mitogen-activated protein kinase (MAPK) pathway and the phosphoinositide 3-kinase (PI3K) pathway are intracellular signaling pathways that are involved in carcinogenesis. Various studies suggest that these pathways are important for the development of liver tumors. To investigate the effect of coactivation of MAPK and PI3K pathways in liver tumorigenesis, we generated three types of mouse models by crossbreeding conditional KrasG12D knockin mice and/or conditional Pten knockout mice with Alb-Cre mice (Alb-Cre KrasG12D/+ Pten flox/flox mice, Alb-Cre KrasG12D/+ Pten flox/+ mice, and Alb-Cre KrasG12D/+ mice). Among the three models, mice carrying liver-specific oncogenic Kras knockin and homozygous Pten deletion (Alb-Cre KrasG12D/+ Pten flox/flox) frequently demonstrated weight loss, abdominal distension, and jaundice with median survival of eight weeks. Macroscopic analysis detected diffuse and firm tumorous lesions mainly in the hepatic hilum. Histological examination of the Alb-Cre KrasG12D/+ Pten flox/flox mice disclosed bile duct hyperplasia at four weeks of age, precancerous lesions of intrahepatic cholangiocarcinoma (ICC) at five weeks of age, and advanced ICC lesions that resemble human ICC at seven weeks of age. On the other hand, the Alb-Cre KrasG12D/+ Pten flox/+ mice, the second model, developed ICCs as well as hepatocellular carcinomas (HCCs), and their average survival was eight months. The third mice model, the Alb-Cre KrasG12D/+ mice, developed HCCs but not ICCs, and survived more than one year.
Immunohistochemical analysis of the ICCs in the Alb-Cre KrasG12D/+ Pten flox/flox mice corroborated the activation of the Mapk and the Pi3k pathways in the tumor cells. These data suggest that activity of Pi3k pathway might dictate the fate determination of hepatotumorigenesis into biliary or hepatocyte cell lineage. In addition, oncogenic Kras expression and homozygous Pten deletion cooperate to induce exclusively ICCs. Since a limited number of mouse ICC models have been reported, the Alb-Cre KrasG12D/+ Pten flox/flox mouse model should be a useful tool for the analysis of molecular mechanism(s) underlying ICC.
Citation Format: Yumi Terakado, Tsuneo Ikenoue, Kiyoshi Yamaguchi, Yoichi Furukawa. Establishment of a novel mouse model of intrahepatic cholangiocarcinoma by liver-specific Kras activation and Pten deletion. [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 652.
Abstract 658: Elucidation of cellular origin of mouse intrahepatic cholangiocarcinoma induced by liver-specific Kras activation and Pten deletion
Authors: Ikenoue Tsuneo , Terakado Yumi , Yamaguchi Kiyoshi , Furukawa Yoichi 
Published: July 2016

Abstract
Regarding the carcinogenesis of intrahepatic cholangiocarcinoma (ICC), growing interest has been focused on its cellular origin, namely cholangiocytes, progenitor cells, or hepatocytes. We have recently established a novel mouse model of ICC by introducing liver-specific Kras activation and Pten deletion using Alb-Cre mice, in which Cre/loxP-mediated gene recombination was achieved in hepatoblasts and mature hepatocytes. To elucidate the cellular origin of ICC in this mouse model, we utilized hepatocyte or cholangiocyte-lineage specific gene engineering using a tamoxifen (TMX) inducible-Cre/loxP system. The mice carrying Kras activation and Pten deletion by Alb-CreERT2 mice with the TMX treatment at eight weeks of age developed hepatocellular carcinoma and hepatocyte dysplasia within three months. Fluorescence microscopy analyses disclosed that the Cre/loxP-mediated gene recombination was specifically observed in hepatocytes in the mice. In contrast, the mice carrying Kras activation and Pten deletion by Alb-CreERT2 mice at ten days of age induced ICC alone within two months. The gene recombination occurred in cholangiocytes as well as hepatocytes. Expectedly, ICCs were developed in the mice carrying cholangiocyte-specific Kras activation and Pten deletion by K19CreERT mice treated with TMX at 8 weeks after birth. TMX-induced, Cre/loxP-mediated recombination was achieved in epithelia of various organs including intrahepatic bile ducts in these mice. These results suggest that the cellular origin of ICC in mice carrying liver-specific Kras activation and Pten deletion was cholangiocytes but not hepatocytes. This mouse model should be useful for better understanding of human ICC of cholangiocyte-origin and the development of new therapeutic strategies for the disease.
Citation Format: Tsuneo Ikenoue, Yumi Terakado, Kiyoshi Yamaguchi, Yoichi Furukawa. Elucidation of cellular origin of mouse intrahepatic cholangiocarcinoma induced by liver-specific Kras activation and Pten deletion. [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 658.
Abstract 663: Factors affecting genome editing using CRISPR/Cas9 in mouse model
Authors: Zheng Qi , Kong Ling-Jie , Jin Huanyu , Li Jinling , Chen-Tsai Ruby Yanru 
Published: July 2016

Abstract
CRISPR/Cas9 joined genome editing family and became a powerful tool for cancer researches. While CRISPR is effective for generating knock-out, knock-in or point mutation animal models, the protocols and success rates to generate CRISPR mouse model vary significantly among different laboratories. We examined a series of parameters for generating various types of mouse models using CRISPR/Cas9. The efficiency and toxicity of gRNA, Cas9 mRNA and donor concentration, insert size and injection methods were thoroughly analyzed. We also evaluated strategies generating conditional knock-out models using CRISPR. The optimized protocols for successful mouse model generation for knock-in, knock-out, point mutation and conditional knock-out using CRISPR will be proposed.
Citation Format: Qi Zheng, Ling-Jie Kong, Huanyu Jin, Jinling Li, Ruby Yanru Chen-Tsai. Factors affecting genome editing using CRISPR/Cas9 in mouse model. [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 663.