bluuebunny/crossref_metadata_2025 huggingface dataset:
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

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

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

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

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

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

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

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.
Abstract 673: The role of MYPT1/2, ASPP2 and MYH9 in invasive lobular carcinoma
Authors: Schipper Koen , de Ruiter Julian , Kas Sjors , Schut Eva , Koudijs Marco , Addams David , Jonkers Jos
Published: July 2016

Invasive lobular carcinoma (ILC) accounts for 10-15% of breast cancer cases in women. One of the characteristics of this type of cancer is the loss of intercellular adhesion which is facilitated by inactivating mutations in E-cadherin. However the loss of E-cadherin alone is not enough to induce ILC. An in vivo Sleeping Beauty insertional mutagenesis screen was performed to identify genes that together with E-cadherin loss contribute to ILC development in mice. In around 85% of the tumors that were analyzed during this screen one of four hits were observed, namely MYPT1, MYPT2, ASPP2 and MYH9. Interestingly these four hits appear to be mutually exclusive indicating a shared mechanism of action.

To investigate how these genes might affect tumorigenesis we first looked at the location of the transposon insertions. This revealed that for MYPT1, MYPT2 and ASPP2 the transposons appeared to localize to a specific region in the gene indicating that these insertions could result in a truncation variant. In the case of MYH9 no clear localization of insertions was found which in combination with the recently published data indicates a loss of function. Northern blot analysis revealed the presence of truncation variants for MYPT1/2 and ASPP2 which for MYPT1 were confirmed to also result in truncated protein variants.

In order to identify the tumorigenic potential of truncated MYPT1 and ASPP2 we overexpressed them in spontaneously immortalized mammary epithelial cell lines HC11 and NMuMG. We are using these cell lines to analyse differences in cell proliferation, anchorage independent growth, sensitivity to apoptosis in vitro and tumor formation in vivo. To further analyse the hits in an E-cadherin negative setting we made use of primary mammary epithelial cells (MECs) isolated from genetically engineered mice with mammary gland specific E-cadherin loss. These MECs normally do not grow past several passages in vitro. However, these MECs when we overexpress truncated MYPT1 or ASPP2, or use shRNA mediated knockdown of MYH9 can be passaged and grown in vitro consistently.

Finally we are generating conditional mouse models with mammary gland specific E-cadherin loss in combination of expression of the truncated variants of MYPT1 or ASPP2, or loss of MYH9. We will to monitor whether these mice are prone to developing ILC.

Citation Format: Koen Schipper, Julian de Ruiter, Sjors Kas, Eva Schut, Marco Koudijs, David Addams, Jos Jonkers. The role of MYPT1/2, ASPP2 and MYH9 in invasive lobular 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 673.
Abstract 68: Neuroblastoma drug response profiles are associated with gene expression profiles
Authors: Bond Jeffrey P. , VanSickle Elizabeth , Dexhemier Thomas S. , Neubig Richard R. , Sholler Giselle L.
Published: July 2016

Background: High risk neuroblastoma patients have a poor response to initial chemotherapy, with 20% unable to achieve a complete response. Genomic studies have identified 6 targeted agents whose pathways have been identified in some high risk neuroblastoma tumors. The low frequency of established driver mutations, along with the variety of molecular mechanisms for cancer pathway dysregulation, suggest development of gene expression-based predictive biomarkers in addition to sequence-based biomarkers. Our platform for developing gene expression-based biomarkers includes primary neuroblastoma cell lines, high throughput quantification of cell survival, exome sequencing, and genome-wide expression profiling.

Methods: Cell survival was quantified for each of thirty primary neuroblastoma cell lines in the presence of each of six drugs (bortezomib, crizotinib, dasatinib, lapatinib, sorafenib, and vorinostat) using high throughput cell survival measurements. Titration curves were based on treatment with vehicle as well as sixteen concentrations of each drug (replicated within and between plates). Summary descriptions of the response of each cell line to each drug were obtained using the four parameter logistic model as well as the area under the curve. Exome sequences were obtained using Illumina HiSeq technology. Genome-wide expression profiles were obtained using Affymetrix GeneChip technology. Inference was based on parametric univariate and non-parametric multivariate tests.

Results: For three of the drugs (bortezomib, dasatinib, and vorinostat) the variation in response between cell lines was statistically significant (p < 0.05; F ≥3) and assay results were reproducible in the context of the differences between cell lines (for the other three drugs the estimated standard deviation of log10 EC50 ≤ 0.2). Genome-wide expression profiles were associated with variation in drug response between cell lines, for example, high PDGFRB expression indicates sensitivity to dasatinib (p < 0.001).

Conclusion: High throughput cell survival measurements provided for identification of predictive biomarkers of neuroblastoma drug sensitivity. These biomarkers are currently being studied in the PEDS-PLAN (Pediatric Precision Laboratory Advanced Neuroblastoma Therapy) clinical trial.

Citation Format: Jeffrey P. Bond, Elizabeth VanSickle, Thomas S. Dexhemier, Richard R. Neubig, Giselle L. Sholler. Neuroblastoma drug response profiles are associated with gene expression profiles. [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 68.
Abstract 681: Using a genome-editing approach for the stepwise establishment of zebrafish models of pediatric high-grade gliomas and MPNSTs
Authors: Oppel Felix , He Shuning , Tao Ting , Zimmerman Mark W. , Durbin Adam D. , Weichert Nina , Ki Dong H. , Look A Thomas
Published: July 2016

By high-throughput sequencing of cancer patient's genomes, driver mutations have recently been described in high-grade pediatric glioma, an aggressive type of brain tumor in children. The most prominent recurrent mutations were found in four genes, namely NF1, TP53, ATRX and H3F3A. NF1 and TP53 are tumor suppressor genes and their loss of function is characteristic for various human malignancies. However, the mechanisms through which precise missense mutations in H3F3A and loss-of-function of ATRX influence tumor biology remain largely unknown. In our project, we employ the genome-editing technology CRISPR-Cas9 to develop a zebrafish model of pediatric high-grade glioma, in order to examine the impact of these mutations on tumor onset and progression. We previously created a zebrafish line that is deficient for tp53 (tp53-/-) and has a loss-of-function mutation in 3 of 4 alleles of nf1 (nf1a+/-, nf1b-/-), which is duplicated in zebrafish. These fish develop high-grade gliomas with low penetrance and malignant peripheral nerve sheath tumors (MPNSTs) with high penetrance. By CRISPR-Cas9 we further incorporated early frameshift mutations of atrx in the background of tp53-/-, nf1a+/-, nf1b-/-. All analyzed fish injected with Cas9-mRNA and CRISPR guide-RNAs (gRNAs) targeting atrx coding sequences showed germline transmission of mutant alleles, of which one third were frameshift mutations. Primary CRISPR-Cas9 atrx-gRNA injected tp53-/-, nf1a+/-, nf1b-/- fertilized eggs developed as mosaics and exhibited increased tumor penetrance and faster onset of MPNSTs and these tumors harbored atrx mutations in 8/10 analyzed samples. This indicates that atrx loss-of-function mutations cooperate with loss of tp53 and nf1 tumor suppressors. Gliomas were not observed in mosaic atrx mutated fish, and these will be reanalyzed in stable atrx mutant lines. Next, we will add previously described missense mutations of h3f3a. To unravel the synergy between oncogenic mutations in atrx and h3f3a in tumor progression, mutant stable lines for both genes will be investigated independently and in a combined manner. The zebrafish model of pediatric high-grade gliomas and MPNSTs that we are developing will be useful to dissect the mechanisms underlying cooperation among driver mutations and for small molecule screens to identify specific inhibitors of cell growth and survival in these malignancies.

Citation Format: Felix Oppel, Shuning He, Ting Tao, Mark W. Zimmerman, Adam D. Durbin, Nina Weichert, Dong H. Ki, A Thomas Look. Using a genome-editing approach for the stepwise establishment of zebrafish models of pediatric high-grade gliomas and MPNSTs. [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 681.
Abstract 686: MNK1 promotes the progression of breast ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC)
Authors: Guo Qianyu , del Rincon Sonia V. , Goncalves Christophe , Miller Wilson H.
Published: July 2016

The mechanism by which breast ductal carcinoma in situ (DCIS) progresses to invasive ductal carcinoma (IDC) is poorly described. Previous studies have revealed that DCIS and IDC share similar genomes and transcriptomes. We hypothesize that one mechanism contributing to the DCIS to IDC conversion involves aberrations in mRNA translation. The Mnk/eIF4E axis has a critical role in promoting the translation of tumor promoting and pro-invasive mRNAs, thus we propose to study whether aberrant activation of this axis promotes the transition of DCIS to IDC. Our model system involved overexpressing constitutively active Mnk1 (caMNK1) in MCF10 variants: (1) MCF10A immortalized mammary epithelial cells and (2) MCFDCIS.com ductal carcinoma in situ cells (termed DCIS cells). Our in vitro results suggest that caMNK1 enables DCIS cells to gain invasive properties. First, overexpression of caMnk1 increased colony formation, larger acinar size in 3D culture, and promoted the migration and invasion of MCF10A and MCFDCIS cells. Second, SEL201, a novel Mnk1/2 inhibitor, can inhibit proliferation, colony formation, 3D acini formation, and the migration and invasion capacity of DCIS-pBABE and -caMNK1 expressing cells. Tumor xenografting in athymic nude mice revealed that caMNK1 facilitates the conversion of DCIS to IDC as the DCIS-caMNK1 expressing tumors progress to IDC, while DCIS-pBABE derived tumors retain DCIS-like structures. Breast tumor samples from patients with DCIS, IDC or mixed DCIS-IDC will also be obtained to examine the levels of phospho-Mnk1 and phospho-eIF4E.

Citation Format: Qianyu Guo, Sonia V. del Rincon, Christophe Goncalves, Wilson H. Miller. MNK1 promotes the progression of breast ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC). [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 686.
Abstract 693: Oncogenic and osteolytic function of histone demethylase NO66 in castration resistant prostate cancer
Authors: Bagheri-Yarmand Rozita , Navone Nora , Wan Xinhai , Logothetis Christopher , Huard Johnny , Sinha Krishna
Published: July 2016

Androgen ablation by pharmacological use or castration is a standard therapy for treating prostate cancer (PCa), however, at the later stage PCa becomes castration-resistant and is metastasized primarily to bones. Epigenetic changes that cause dysregulated gene expression during progression and metastasis of androgen-independent prostate cancer are not clearly understood. We previously reported that the jumonji C domain-containing NO66 is a histone demethylase specific for H3K4me3 and H3K36me3, which negatively regulates osteoblast differentiation and bone formation. By analyzing a multi-cancer tissue microarray, the serendipitous observation was made that NO66 is overexpressed in a variety of cancers. Our objective was to examine NO66's role in the pathogenesis of prostate cancer and bone metastasis.

Our findings indicate that NO66 knockdown resulted in significant decreases in the proliferation and invasiveness of prostate cancer cells (PC3 and DU145) while overexpressing NO66 promoted their proliferation and invasion. Furthermore, NO66 depletion resulted in a two- to three fold decrease in colony formation and anchorage-independent growth of PCa cells suggesting an oncogenic function of NO66. RNAseq analysis comparing between control and NO66-depleted DU-145 cells revealed a repertoire of differentially expressed genes. NO66 up-regulates genes with proliferative activity such as chemokine CXCL5, growth factor IGFBP5, IL6, WNT and MAPK4 pathway, which are known to drive PCa proliferation. Furthermore, ChIP sequencing for NO66 and H3K9AC in NO66-overexpressing PC3 cells revealed that interaction sites of NO66 overlap with peaks of H3K9AC in majority of the genes. These data clearly indicate that NO66 activates a cohort of genes which are involved in tumor progression and metastasis. Strikingly, implanting NO66-overexpressing prostate cancer cells into the femur of SCID male mice caused massive femoral bone loss after three weeks, which suggests that NO66 causes osteolytic bone lesions similar to the clinico-pathological features of advanced PCa. Finally, analysis of tumor xenografts derived from patients by immunohistochemistry showed high expression of NO66 in high grade advanced PCa compared to low grade and hyperplasia suggesting that NO66 may be used as a biomarker to distinguish aggressive prostate cancer from indolent stage. Our data suggest that NO66 plays an important role in cell proliferation, cell invasion and bone metastasis of prostate cancer.

Citation Format: Rozita Bagheri-Yarmand, Nora Navone, Xinhai Wan, Christopher Logothetis, Johnny Huard, Krishna Sinha. Oncogenic and osteolytic function of histone demethylase NO66 in castration resistant prostate 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 693.
Abstract 695: SHP-2-upregulated ZEB1 is important for PDGFRá-driven glioma epithelial-mesenchymal transition and invasion in mice and humans
Authors: Zhang Lei , Zhang Weiwei , Li Yanxin , Li Zuoqing , Wang Yinfang , Song Lina , Lv Deguan , Nakano Ichiro , Hu Bo , Cheng Shi-Yuan , Feng Haizhong
Published: July 2016

A critical clinical challenge in glioblastoma therapy is robust tumor growth and invasion driven by aberrant activation of oncogenic tyrosine receptor kinases (RTKs) including PDGFRá. Here, we report that a SHP-2-upregulated epithelial-mesenchymal transition (EMT)-inducer, ZEB1 is important for PDGFRá-driven glioma EMT, invasion and glioma stem cell renewal in mice and humans. ZEB1 and activated PDGFRá were co-expressed in invasive regions of mouse glioma xenografts and human clinical glioma specimens. Glioma patients with high levels of both p-PDGFRá and ZEB1 had significantly shorter overall survival compared with those with low expression of p-PDGFRá and ZEB1. Knockdown of ZEB1 inhibited PDGF-A/PDGFRá-stimulated glioma EMT, tumor growth, invasion and glioma stem cell renewal. PDGFRá mutant deficient of SHP-2 binding (PDGFRá-F720) or PI3K binding (PDGFRá-F731/42), knockdown of SHP-2 or treatments of pharmacological inhibitor for PDGFRá-signaling effectors attenuated PDGF-A/PDGFRá-stimulated ZEB1 expression, cell migration and glioma stem cell proliferation. Importantly, SHP-2 acts together with PI3K/Akt to regulate a ZEB1-miR-200 feedback loop in PDGFRá-driven gliomas. Together, our findings uncover a new pathway in which ZEB1 functions as a key regulator for PDGFRá-driven glioma EMT, invasion and glioma stem cell renewal, suggesting that ZEB1 as a potential therapeutic target for human gliomas with high PDGFRá activation.

Citation Format: Lei Zhang, Weiwei Zhang, Yanxin Li, Zuoqing Li, Yinfang Wang, Lina Song, Deguan Lv, Ichiro Nakano, Bo Hu, Shi-Yuan Cheng, Haizhong Feng. SHP-2-upregulated ZEB1 is important for PDGFRá-driven glioma epithelial-mesenchymal transition and invasion in mice and humans. [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 695.
Abstract 719: ICAM3 and CCL16, inflammation genes screened by high-throughput siRNA library, govern cancer stemness in malignant tumors
Authors: Shen Wenzhi , Xie Junling , Du Renle , Jiang Shan , Luo Xiaohe , He Huiwen , Xiang Rong , Luo Yunping
Published: July 2016

Cancer stem cells (CSCs) have the ability of both self-renewal and differentiation, were thought to be the main root of tumorigenesis, metastasis, recurrence and drug resistance. Current studies have focused on the maintenance of cancer stem cell abilities. Moreover, existing studies have shown that tumor associated inflammation microenvironment (TIM) play a pivotal role in different stages of tumor development. The linkage between inflammation and tumor stemness has been established. However, the evidence of TIM function on maintaining and nourishing CSCs properties remains insufficient. Here, we performed a high throughput siRNA interference platform to systemically screen out the inflammatory genes that regulate tumor stemness via OCT4 promoter linked reporter system in HMLE-Snail cells. 10 candidates were screened out which knockdown could both down-regulate OCT4 expression and decrease ALDH+ subpopulation. Furthermore, we validated the function of genes ICAM3 and CCL16 and found that knockdown ICAM3 or CCL16 inhibit cell migration, sphere formation in 231, A549 and HepG2 cell lines. We also found ICAM3 or CCL16 deficiency decrease side population and reduce chemo-resistance. In addition, mice bearing ICAM3 or CCL16 knockdown breast cancer cells develop smaller tumors and less lung metastases verse controls. Taken together, our study demonstrated that the inflammation related genes ICAM3 and CCL16 govern cancer cell migration and stemness in malignant tumors in vitro and in vivo, and that will provide new possible targets to CSCs for cancer therapy.

Citation Format: Wenzhi Shen, Junling Xie, Renle Du, Shan Jiang, Xiaohe Luo, Huiwen He, Rong Xiang, Yunping Luo. ICAM3 and CCL16, inflammation genes screened by high-throughput siRNA library, govern cancer stemness in malignant tumors. [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 719.
Abstract 72: Therapeutic compound discovery targeting a recurrent glioblastoma (GBM) phenotype using LINCS compounds via QUADrATiC analyses
Authors: Al Rashid Shahnaz T. , O’Reilly Paul , Dunne Philip D. , Alderdice Matthew , Flannery Thomas J. , Prise Kevin M. , Zhang Shu-Dong , McArt Darragh G.
Published: July 2016

Glioblastoma (GBM) is the most prevalent and aggressive of malignant primary brain tumours. Despite multi-modal therapy, including surgical resection, radiotherapy and chemotherapy, GBM continues to be uniformly lethal, with a median survival of 12-18 months from the time of diagnosis. The resistance to therapy and ultimate recurrence is thought to originate from a sub-population of cells residing in the initial tumour, identified as glioma-initiating or glioma-stem cells (GSC). These GSC have been shown to be intrinsically more invasive, with enhanced repair and drug-efflux pathways mediating resistance to chemo-radiation therapies. The purpose of this study was to use and validate a novel connectivity mapping procedure to identify candidate compounds targeting therapy-resistant and recurrent GBM. This procedure was developed by O’Reilly and colleagues (“QUADrATiC: A Scalable Approach to Repurposing FDA-approved therapeutics”, under review in Bioinformatics), and in this study, was performed on publicly available NCBI GEO microarray datasets.

Based on the cancer stem cell theory whereby the resistant GSCs repopulate and are enriched in recurrent tumours, primary and recurrent GBM gene expression profiles were compared and the connections between the identified gene lists and drug responses were mapped. To validate that this connectivity mapping procedure is reliable at identifying effective treatments to target therapeutic resistance and recurrence, we tested one of the top ten compounds identified, rosiglitazone (rosi), a ligand of the peroxisome proliferator activated receptor gamma (PPARγ). The efficacy of rosi alone and in combination with the current standard of care therapies, temozolomide and radiation, at killing GSC was assessed in vitro using the clonogenic survival assay. Our data indicated that rosi significantly reduced survival of both non-stem glioma cells and GSC, alone and in combination with radiation and temozolomide. The p53 pathway was identified as a pathway by which rosi may induce permanent cell cycle arrest and cell death. Further work is ongoing to fully elucidate the downstream mechanisms of cell death. This work is proof-of-principle that the current connectivity mapping procedure can be instrumental in identifying more targeted therapeutic interventions for GBM resistance and prevent recurrence.

Citation Format: Shahnaz T. Al Rashid, Paul O’Reilly, Philip D. Dunne, Matthew Alderdice, Thomas J. Flannery, Kevin M. Prise, Shu-Dong Zhang, Darragh G. McArt. Therapeutic compound discovery targeting a recurrent glioblastoma (GBM) phenotype using LINCS compounds via QUADrATiC analyses. [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 72.
Abstract 720: Novel prognostic stromal subtypes in triple-negative breast cancer
Authors: Thompson Crista , Saleh Sadiq M. , Bertos Nicholas , Gigoux Mathieu , Gruosso Tina , Souleimanova Margarita , Zhao Hong , Hallett Michael T. , Park Morag
Published: July 2016

Breast cancer is a heterogeneous disease in terms of presentation, morphology, molecular profile and response to therapy. Gene expression profiling has identified intrinsic molecular subtypes that are associated with clinical markers (ER, PR, HER2) as well as prognosis and survival. However, it is well established that the intrinsic molecular profiles of breast tumors are not sufficient to perfectly predict disease outcome. Increasing evidence indicates that characteristics of the breast stroma influence tumor progression and response to therapy. Previous work in our lab has demonstrated that gene expression signatures in human stroma can predict outcome of breast cancer patients independently of clinical parameters and molecular subtypes. In this study, we expand our findings by focusing on a previously underrepresented subset of breast tumors that have no detectable ER, PR or HER2 (termed Triple-Negative, TN). TN tumors, which represent approximately 15% of all breast cancers, are typically associated with poor outcome. However, the contribution of the stroma to the underlying heterogeneity of TN breast cancer and its corresponding influence on therapeutic response is not well understood. To address this, we isolated TN tumor epithelial and stromal tissues by laser capture microdissection and subjected them to gene expression profiling. Class discovery revealed distinct gene-clusters (stromal properties) which are associated with prognosis in TNBC whole tumor samples. Analysis of the genes comprising each stromal property suggests that the properties primarily represent the prevalence of distinct cell types, namely T cells, B cells, activated fibroblasts, and myoepithelial cells. Importantly, these properties are not mutually exclusive, i.e. some tumors are associated with multiple stromal properties. While confirming the heterogeneity of TN-associated stroma, this also indicates that a multi-parameter classification better reflects the true nature of the tumor microenvironment. This project provides the first integrated in-depth analysis of the contribution of tumor stromal processes to TN disease heterogeneity, and positions the tumor microenvironment for therapeutic intervention.

Citation Format: Crista Thompson, Sadiq M. Saleh, Nicholas Bertos, Mathieu Gigoux, Tina Gruosso, Margarita Souleimanova, Hong Zhao, Michael T. Hallett, Morag Park. Novel prognostic stromal subtypes in triple-negative 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 720.
Abstract 721: Opposite roles of CD11b+ myeloid cells in primary and recurrent brain tumors
Authors: Sheng-Yen Wu
Published: July 2016

Malignant glioma is one of the toughest tumors to be treated at present due to the complexity of the tumor microenvironment and the intrinsic resistant to irradiation. Previous studies have shown that CD11b+ myleoid cells play essential role in brain tumor recurrence following radiation therapy. Here we used the CD11b-diphtheria toxin receptor (CD11b-DTR) transgenic mouse model to evaluate the role of CD11b+ myleoid cells in TK/GCV suicide gene therapy for brain tumor in a murine astrocytomal tumor model, ALTS1C1-TK. Using this transgenic mouse model, the depletion of peritoneal macrophages (CD11b+F4/80+) and blood monocyte (CD11b+Ly6G−Ly6c−) could be achieved after two injections of DT, but the neutrophil (CD11b+Gr-1+) were increased transiently. Results found that the depletion of CD11b+ myleoid cells enhanced the efficacy of TK/GCV therapy as shown by the increase of median surviving time of GCV-treated ALTS1C1-TK tumor-bearing mice from 30.6 days to 37.5 days. Interestingly, the depletion of CD11b+ myleoid cells enhanced tumor growth as shown that tumor-bearing mice had shorter surviving days and larger tumor size after receiving two doses of DT injections compared to the control PBS group (22.4 days vs 25.0 days, respectively). This indicates that depleted CD11b+ myleoid cells might have anti-tumor function for control tumor, but have pro-tumor activity during TK/GCV therapy. This study also found the effect of CD11b+ myleoid cells was only seen when the depletion was performed during the administration of GCV, but not before or after GCV treatment. Collectively, above findings suggest that CD11b+ myleoid cells might have different roles on primary and recurrent brain tumors.

Citation Format: Wu Sheng-Yen. Opposite roles of CD11b+ myeloid cells in primary and recurrent brain tumors. [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 721.
Abstract 725: HMGB1-mediated RAGE activation mechanism in M2 macrophages
Authors: Rojas Armando , Araya Paulina , Romero Jacqueline , Delgado-López Fernando , Pérez-Castro Ramón , González Ileana , Añazco Carolina , Morales Erik , Llanos Jorge , Vidal-Vanaclocha Fernando
Published: July 2016

Tumor-associated macrophages display M2 phenotype and promote immunosuppression, tumor growth, angiogenesis and metastasis. We have previously reported that M2 macrophages express the receptor of advanced glycation end-products (RAGE) and their protumoral activities enhanced by HMGB1, a RAGE ligand highly abundant at the cancer microenvironment. However, RAGE is highly expressed by M1 macrophages and it is unknown if proinflammatory and antitumor cytotoxicity of M1 macrophages become skewed from classical antitumor activities to a tumor permissive profile when M2 macrophages are induced by HMGB1.

In order to clarify whether RAGE contributes to inflammatory response inhibition in M2 macrophages, we look at some microRNAs, a tolerance-like state as well as to some epigenetic changes. Polarized M1 and M2 macrophages were derived from wild-type and RAGE-targeted knock-down THP-1 cells and treated with HMGB1 at different times. NFkB activation was assessed by p65, IKBalpha western blots, and p50 and p65 Transcription Factor Assay. Additionally, we analyzed changes of chromatin activity related to HMGB1 treatment by quantitative chromatin immunoprecipitation (ChIP) with antibodies against acetylated or methylated histone H3 at the IL-10 promoter.

We showed that RAGE activation by HMGB1 did not produce NFkB activation, as assessed by either IKB alpha and phospho p65 western blots or by p65 and p50 trascription factor assays. In addition, HMGB1 did not modify M2 macrophage expression profile of relevant microRNAs for macrophage polarization such as miR-155, miR-21 and miR-125b. However, ChIP analysis showed that HMGB1 induced a transcription-prone histone modification at the IL-10 promoter in M2 macrophages and this epigenetic imprinting correlated with HMGB1-induced IL-10 production, leading to immunosupression. These results demonstrate that RAGE activation on M2 macrophages did not induce NFkB activation and suggest that epigenetic changes are a major mechanism by which activities described for M1 macrophages upon RAGE activation have become skewed during macrophage polarization.

Citation Format: Armando Rojas, Paulina Araya, Jacqueline Romero, Fernando Delgado-López, Ramón Pérez-Castro, Ileana González, Carolina Añazco, Erik Morales, Jorge Llanos, Fernando Vidal-Vanaclocha. HMGB1-mediated RAGE activation mechanism in M2 macrophages. [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 725.