bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3063: Molecular characterization of individual circulating tumor cells by RNA sequencing
Authors: Patel Jaymala , Foulk Brad , Bhargava Vipul , Smirnov Denis A.
Published: May 2015

Background: Numbers of circulating tumor cells (CTC) measured with the CellSearch system in patients with metastatic carcinomas are associated with poor survival. Such association with clinical outcome suggests that CTCs play an important role in metastatic process. Molecular characterization of CTCs can provide important insights into phenotypic characteristics of cells necessary to participate in this process. Moreover, detailed molecular characterization of CTCs at various points of the disease course may be useful in understanding of evolution of tumors and aid in selection of optimal therapeutic regimens. Like most other CTC isolation instruments CellSearch is a CTC-enrichment device that efficiently captures CTCs while also retaining few thousands non-specifically captured white blood cells. To obtain deep molecular profiles of CTCs isolation of single cells is required and development of specialized next-generation sequencing protocols is necessary.

Method/Results: We developed a workflow to enable isolation of single CTCs or pools of few CTCs following enrichment with the FDA approved CellSearch platform. Experiments with cancer cell lines spiked into donor blood showed 80% cellular recovery from blood using CellSearch and subsequent 40-50% capture of individual cancer cells using Fluidigm C1 Single Cell system. Following analysis of gene expression via RNAseq expression of ∼5000 genes can be detected in isolated tumor cells. We will discuss reagents and molecular approaches utilized to ensure efficient isolation and quality assessment of nucleic acids recovered from isolated tumor cells. We will discuss impact of sample preservation has on transcriptome profiling. We will share insights into prostate and lung cancers that one can gain from such analysis.

Conclusion: Characterization of individual CTCs by RNA sequencing is a promising approach to characterize metastatic processes and tumor evolution.

Citation Format: Jaymala Patel, Brad Foulk, Vipul Bhargava, Denis A. Smirnov. Molecular characterization of individual circulating tumor cells by RNA sequencing. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3063. doi:10.1158/1538-7445.AM2014-3063
Abstract 3078: Isolation and in vitro culturing of human circulating tumor cells
Authors: Bobek Vladimir , Kolostova Katarina
Published: May 2015

The CTC-enrichment based on the size of the cells, is addressing the problem of reduced EpCAM expression, but filtration shows relatively low sensitivity and specificity, because small CTCs may escape of the detection, whereas big leukocytes may contaminate population of CTCs Isolation by size of epithelial tumor cells is based on use of filters, which preferentially capture tumor cells bigger than 8 uM.

Our filtration device (MetaCell®, Metacell s.r.o., Ostrava, Czech Republic) isolates tumor cells by size from a blood sample up to 6 ml. Moreover immunocytochemistry and FISH assays can be performed on the filter. The protocol enables to isolate viable cells, allowing further in vitro experiments. Furthermore, tumor cells can be isolated without using an antibody-based approach, suggesting that the device can be used for the isolation of a large spectrum of tumor cells, including cell of non-epithelial origin.

We presented the successful isolation and cultivation of CTC/DTC of the following cancer: NSCLC, mesothelioma, oesophageal cancer, colorectal cancer, gastric cancer, pancreatic cancer, leiomyosarcoma, prostate cancer, malignant melanoma and renal cancer.

Citation Format: Vladimir Bobek, Katarina Kolostova. Isolation and in vitro culturing of human circulating tumor cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3078. doi:10.1158/1538-7445.AM2014-3078
Abstract 310: Identification of secreted proteins that reflect intracellular autophagy dynamics in melanoma
Authors: Akl Adam , Xu Xiaowei , Piao Shengfu , Amaravadi Ravi K. , Speicher David W.
Published: May 2015

Autophagy, a catabolic process that maintains cellular homeostasis, is a primary therapy resistance mechanism in cancer. Emerging studies indicate that autophagy is involved in secretion through non-classical mechanisms. However, the effects of autophagy-mediated secretion on the tumor microenvironment are poorly understood. Further, while autophagy is a potential cancer therapeutic target, efforts to inhibit autophagy in clinical trials have been hampered by sub-optimal methods to quantitatively monitor tumor autophagy levels. Here, we leveraged the recent findings that autophagy is implicated in non-classical secretion to identify secreted proteins associated with autophagy in melanoma. The conditioned media of low autophagy WM793 melanoma cells was compared to its highly autophagic metastatic derivative, 1205Lu, using quantitative proteomics. These comparisons identified secreted proteins associated with high autophagy, such as IL-1β, IL-8, LIF, FAM3C, and DKK3, which have previously been implicated in tumorigenesis. These proteins were shown to be elevated in supernatants of an independent panel of high autophagy melanoma cell lines compared with low autophagy cells, suggesting a mechanistic link between autophagy level and secretion of these proteins. In addition, secretion of these proteins increased when melanoma cells with intrinsically low autophagy levels were treated with an autophagy-inducing tat-Beclin 1 peptide. Further supporting a mechanistic link, Atg7 silencing in an intrinsically high autophagy cell line resulted in significantly decreased secretion. Finally, serum collected from untreated metastatic melanoma patients with high tumor autophagy levels exhibited higher levels of these proteins than serum from low autophagy patients. These results suggest that autophagy-related secretion affects the tumor microenvironment and that measurement of extracellular autophagy-associated secreted proteins in tumors and in plasma can serve as surrogates for intracellular autophagy dynamics in tumor cells.

Citation Format: Adam Akl, Xiaowei Xu, Shengfu Piao, Ravi K. Amaravadi, David W. Speicher. Identification of secreted proteins that reflect intracellular autophagy dynamics in melanoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 310. doi:10.1158/1538-7445.AM2014-310
Abstract 3101: Precise matching of medulloblastoma mouse models to their human counterparts
Authors: Pöschl Julia , Stark Sebastian , Neumann Philipp , Gröbner Susanne , Jones David , Northcott Paul , Lichter Peter , Pfister Stefan , Kool Marcel , Schüller Ulrich
Published: May 2015

Mouse models are indispensible tools to study the biology of a tumor and to perform preclinical treatment studies. Medulloblastoma is a very heterogeneous disease with some subgroups being associated with very poor clinical outcome. A lot of effort has been put into the generation of appropriate mouse models. The usage of these models, however, has become debatable with the advances in human medulloblastoma subtyping. In order to precisely characterize the available variety of mouse models we matched genetics and gene expression patterns of 140 murine and 423 human medulloblastomas. Ctnnb1 mutated mouse tumors significantly matched to human WNT medulloblastoma using agreement of differential gene expression (ADGEX) and k-means clustering analysis. Mouse models that have been generated based on Myc or Mycn alterations variably matched to the human Group 3 medulloblastoma depending on the model and the mathematical algorithm, but none of the available models matched to Group 4 tumors. Interestingly, models with Ptch1 or Smo mutations selectively matched to SHH medulloblastomas of adulthood, but the genetic make-up was not the main determinant of the age-dependent gene expression fingerprint. We emphasize important chances and drawbacks of current medulloblastoma models and suggest that the phenotype of medulloblastoma subtypes is orchestrated by distinct combinations of genetics, epigenetics, and cells of origin.

Citation Format: Julia Pöschl, Sebastian Stark, Philipp Neumann, Susanne Gröbner, David Jones, Paul Northcott, Peter Lichter, Stefan Pfister, Marcel Kool, Ulrich Schüller. Precise matching of medulloblastoma mouse models to their human counterparts. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3101. doi:10.1158/1538-7445.AM2014-3101
Abstract 3104: PAX3-FOXO1 increases fibroblast reprogramming efficiency and drives self-renewal in alveolar rhabdomyosarcoma
Authors: Schafer Beat W. , Casanova Elisa , Satheesha Sampoorna , Müller Melanie , Cinelli Paolo , Cinelli Paolo
Published: May 2015

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Most alveolar (ARMS) cases bear the chimeric transcription factor PAX3/7-FOXO1 and have a worse prognosis compared to the embryonal subtype (ERMS). Previous studies identified a CSC subpopulation in ERMS but no study looked at the translocation positive RMS so far. This knowledge would be important to guide future treatment strategies. Here, we show that no CSC subpopulation can be detected in ARMS by a range of common methods. Furthermore, in vivo limiting dilution experiments confirmed that even 100 ARMS cells are sufficient to generate tumors in NOD/SCID mice. Thus, we hypothesized that ARMS follows a clonal evolution model. In line with this hypothesis, the stem cell factors Nanog, Oct4, and Sox2 were homogeneously expressed in ARMS cell lines and genetic loss- or gain-of-function experiments did not change cellular physiology unlike silencing of PAX3-FOXO1 which caused cell cycle arrest and apoptosis. Based on these observations we then investigated whether PAX3-FOXO1 itself can act as stem cell gene. For this we reprogrammed human fibroblast with the known factors Oct4, Sox2, Klf4, and c-Myc in presence or absence of PAX3-FOXO1. Interestingly, the presence of the fusion protein significantly increased the reprogramming efficiency leading to the generation of almost double the number of colonies of induced pluripotent stem cells (iPSC) than the control cells. PAX3-FOXO1 iPSC expressed the core stem cell genes as well as the PAX3-FOXO1 target gene AP2beta. Strikingly, PAX3-FOXO1 iPSC showed reduced ability to differentiate in vitro into all three germ layers. Ongoing experiments aim at confirming these results also in vivo by generation of teratomas. In conclusion and in contrary to ERMS, fusion protein positive ARMS seem to follow the clonal evolution model in which each clone has tumorigenic potential. Moreover, PAX3-FOXO1 is the critical factor for ARMS self-renewal and a general inhibitor of differentiation.

Citation Format: Beat W. Schafer, Elisa Casanova, Sampoorna Satheesha, Melanie Müller, Paolo Cinelli, Paolo Cinelli. PAX3-FOXO1 increases fibroblast reprogramming efficiency and drives self-renewal in alveolar rhabdomyosarcoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3104. doi:10.1158/1538-7445.AM2014-3104
Abstract 3106: The HoxD locus: Its contribution to the osteogenic phenotype and malignancy of Ewing sarcoma
Authors: Heyking Kristina von , Roth Laura , Ertl Miriam , Burdach Stefan , Richter Günther H.
Published: May 2015

Ewing sarcoma (ES), an osteogenic malignancy that mainly affects children and young adults, is characterized by early metastasis to lung and bone. Microarray analysis revealed genes of the posterior HoxD locus to be clearly over-expressed in primary ES. Especially, genes such as HOXD10, HOXD11 and HOXD13 normally involved in bone formation and ossification pattern of bones are significantly over-expressed in ES. The expression of HOXD10, HOXD11 or HOXD13 seems not to be influenced via ES pathognomonic ews/ets translocations, since RNA interference of EWS-FLI1 or its over-expression in mesenchymal stem cells did not influence posterior HOXD expression. However, RNA knock down of the dickkopf 2 homolog DKK2, a canonical WNT/beta-catenin agonist and critical mediator of osteolytic tumor growth in ES (Hauer et al., 2013) resulted in a significant suppression of HOXD11 and HOXD13 expression. Subsequently, RNA interference of individual HoxD genes demonstrated posterior HOXD genes to suppress neuronal differentiation potential of ES. Furthermore, HOXD10, HOXD11 and HOXD13 were found to be critical for chondrogenic, however not for osteogenic differentiation, but promote the expression of bone-associated genes such as RUNX2. HOXD11 and HOXD13 further promote in vitro proliferation and contact independent growth of ES. Similarly, in vitro invasiveness of ES lines was dependent on HOXD10, HOXD11 as well as HOXD13 and seemed mediated via Matrix Metalloproteinase 1 (MMP1), since down-regulation of individual HoxD genes likewise resulted in a suppression of MMP1 expression. In line with these observations, knock down of HOXD11 or HOXD13 significantly suppressed metastasis in a xeno-transplant model in immune deficient Rag2-/-gammaC-/- mice. These data overall provide evidence that posterior HOXD genes are critical mediators of the differentiation phenotype of ES concurrently promoting their metastasis potential.

Reference:

Hauer K, Julia Calzada-Wack J, Steiger K, Grunewald TG, Baumhoer D, Plehm S, Buch T, Prazeres da Costa O, Esposito I, Burdach S, Richter GHS (2013). DKK2 mediates osteolysis, invasiveness and metastatic spread in Ewing sarcoma. Cancer Res. 73(2): 967-77.

Citation Format: Kristina von Heyking, Laura Roth, Miriam Ertl, Stefan Burdach, Günther H. Richter. The HoxD locus: Its contribution to the osteogenic phenotype and malignancy of Ewing sarcoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3106. doi:10.1158/1538-7445.AM2014-3106
Abstract 3108: Copy number variation analysis in neuroblastoma through next generation sequencing data and SNP-microarray
Authors: Fransson Susanne , Östenssson Malin , Djos Anna , Javanmardi Niloufar , Kogner Per , Martinsson Tommy
Published: May 2015

The childhood cancer neuroblastoma show high degree of clinical heterogeneity ranging from aggressive tumors with fatal outcome to cases of spontaneous regression. Analysis of recurrent chromosomal aberrations such as losses of 1p, 11q, gain of 1q, 17q and/or MYCN amplification are currently used for patient stratification and definition of therapeutic strategy. Different analysis techniques for detection of segmental abnormalities include FISH, CGH-microarrays or multiplex ligation-dependent probe amplification (MLPA). However, as next-generation sequencing (NGS) becomes available for clinical use, this could also be used for assessment of copy number alterations simultaneously with mutational analysis. In this study we compare genomic profiles generated through the bioinformatical tool Control-FREEC on Exome sequencing data with profiles generated from Affymetrix 250K or 50K SNP-microarrays on 20 NB tumors of different stages. Exome sequencing was performed by paired-end sequencing on Illumina instrumentation after DNA enrichment with Agilent SureSelect All Exome. The sequencing were performed at three separate occasions with a median raw coverage of 91X, 127X and 340X respectively. Neuroblastoma tumors were normalized with either corresponding constitutional DNA or normal control DNA for NGS data while microarrays were normalized against healthy control DNA. Gross genomic changes were extracted through visual and/or ratio inspection from 20 neuroblastoma tumors. A total of 128 larger segmental aberrations and 63 numerical aneuploidies were detected trough SNP-microarrays. Discrepancies between the two methods were detected in three cases; one segmental loss detected through microarray could not be confirmed by NGS-profiling, one smaller deletion were only detected through NGS-profiling and in one instance both methods indicated 2p-gain but at different positions. Otherwise the results were concordant. Furthermore, through exome sequencing data were we also able to detect ALK mutations in four patients, ATRX-deletions in two patients and chromothripsis of chromosome 5 in one patient. In conclusion, exome sequencing could be used as a new diagnostic tool for neuroblastoma tumors combining mutational screening with detection of common chromosomal and segmental aneuploidies such as 2p-gain, 17q-gain, 11q-deletion as well as MYCN-amplification and alterations of ALK or ATRX.

Citation Format: Susanne Fransson, Malin Östenssson, Anna Djos, Niloufar Javanmardi, Per Kogner, Tommy Martinsson. Copy number variation analysis in neuroblastoma through next generation sequencing data and SNP-microarray. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3108. doi:10.1158/1538-7445.AM2014-3108
Abstract 311: Autophagic pathways and activity in breast and pancreatic cancer
Authors: Egilsson Mar , Thoroddsen Ulfur , Jonasson Jon Gunnlaugur , Ogmundsdottir Margret Helga , Ögmundsdottir Helga M.
Published: May 2015

Autophagy is induced as a homeostatic process by energy shortage and cellular stress but can also be influenced by drugs. Poor autophagic function contributes to carcinogenesis but autophagy also promotes survival of tumours. We performed immunohistochemistry for LC3, pAMP kinase, p62, p53 on 15 samples from breast cancer and 14 from pancreatic cancer. P53 mutations were detected using CDGE. Autophagy was active (LC3 puncta in ≥ 30% of cells) in 5/15 breast carcinomas and 7/14 pancreatic carcinomas. This was associated with energy shortage (strong pAMP kinase expression) in 4/5 in breast samples and 5/7 in those from pancreas, often clearly colocalized when expression was variable. In contrast, activation of AMP kinase was not associated with autophagy in 4/8 breast and 2/7 pancreatic cancers. In 2/5 breast samples and 2/7 from pancreas impaired autophagic flux was suggested by absent cytoplasmic LC3 staining and strong expression of p62. Nuclear expression of p53 was seen in 10/15 breast cancer samples and of those 3 were mutated, 11/14 pancreas samples showed nuclear staining for p53, none mutated. Three out of 7 non-mutated p53-protein-positive breast cancers had low autophagy. Autophagy was thus often associated with energy shortage, can clearly also be induced by other pathways, but p53 appears not to be involved. Lack of autophagy in the presence of energy shortage suggests defects in induction. There is, therefore, considerable variability in autophagic pathways and activity in cancer and this will have to be taken into account when considering therapeutic intervention in the autophagic process.

Citation Format: Mar Egilsson, Ulfur Thoroddsen, Jon Gunnlaugur Jonasson, Margret Helga Ogmundsdottir, Helga M. Ögmundsdottir. Autophagic pathways and activity in breast and pancreatic cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 311. doi:10.1158/1538-7445.AM2014-311
Abstract 3111: Identification of genes that frequently exhibit copy number alterations in pediatric ependymomas
Authors: Hamm Christopher A. , Costa Fabricio F. , Bischof Jared M. , Vanin Elio F. , Bonaldo Maria de Fatima , Iannaccone Steve , Rajaram Veena , George David , Tomita Tadanori , Goldman Stewart , Jennings Lawrence J. , Gilbertson Richard J. , Soares Marcelo B.
Published: May 2015

Ependymoma is a common pediatric central nervous system (CNS) tumor that is believed to originate from ependymal cells located in the lining of ventricular surfaces in the brain. Ependymoma presents great challenges in treatment despite the advances in neurosurgical techniques and adjuvant therapy. To uncover protein interaction networks that are consistently compromised in pediatric ependymomas, we conducted a computational analysis of the array CGH data derived from 203 primary ependymomas from the St. Jude Children's Research Hospital cohort, published by Johnson and colleagues [Nature, 466(7306):632-6, 2010]. The analysis (Bischof and Soares, manuscript in preparation) revealed 15 regions of copy number variation (CNVs) that occurred in at least 50% of the tumors. Peak genes from these CNVs were selected for a confirmatory study using DNA digital PCR assays with an independent ependymoma cohort from the Ann & Robert H Lurie Children's Hospital of Chicago. In this study, all ependymomas displayed copy number alterations in at least one of the 10 genes tested by digital PCR. The digital PCR data sheds light into the biology of ependymomas as the 10 candidate genes have a wide range of cellular functions; from neural stem cell development, to tumor suppression, to the pathogenesis of glioblastoma. In conclusion, we have confirmed that the occurrence of DNA copy number variation in these 10 genes prevail in ependymomas. Indeed, all tumors exhibited at least one of these CNVs. These alterations provide insights into the protein interaction networks underlying development of ependymomas while uncovering targets that may be exploited for therapeutic intervention.

Citation Format: Christopher A. Hamm, Fabricio F. Costa, Jared M. Bischof, Elio F. Vanin, Maria de Fatima Bonaldo, Steve Iannaccone, Veena Rajaram, David George, Tadanori Tomita, Stewart Goldman, Lawrence J. Jennings, Richard J. Gilbertson, Marcelo B. Soares. Identification of genes that frequently exhibit copy number alterations in pediatric ependymomas. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3111. doi:10.1158/1538-7445.AM2014-3111
Abstract 3115: Calcitriol effects on breast cancer tumorgrafts
Authors: Katayama Maria Lucia Hirata , Fonseca-Filho Victor Celso N. , Lyra Eduardo Carneiro , Maria Durvanei Augusto , Basso Ricardo Alves , Nonogaki Suely , Guerra Juliana Mariotti , Maistro Simone , Goes João Carlos Sampaio , Folgueira Maria Aparecida A. Koike
Published: May 2015

Antiproliferative effects of calcitriol were mainly detected in breast carcinoma lineages exposed in vitro to high hormone concentrations, which is associated with hypercalcemia in human beings. To test the hypothesis that intra-tumoral administration of calcitriol would allow higher tissue concentration of the hormone and activation of the genomic pathway, a tumorgraft model, that more closely reproduces the molecular characteristics of the primary tumor, was established. Freshly collected breast cancer samples from 14 patients were ortothopically grafted in nude mice. On the sixth week after xenografting, no palpable nodules were detected in mice implanted with 6 samples. Intra-tumoral weekly injections of calcitriol 0.06 μg (dose that allow peak serum calcitriol levels in the predicted therapeutic range) or vehicle were initiated and maintained for six weeks in mice xenografted with another 8 samples. Tumorgraft proliferation, apoptosis and target genes expression were evaluated through immnunohistochemistry reactions or RT-PCR. At the end of the experiment, tumorgrafts originated from three samples were available for analysis. VDR expression was detected in these samples as well as a trend towards higher expression of CYP24A1 mRNA (10-18 fold) in calcitriol treated samples, indicating that the genomic pathway was induced. A high proliferative index, evaluated by Ki67 expression, was observed, however, neither differences in the expression of proliferation markers (BrdU incorporation, Ki67 and CDKN1B expression) nor in apoptosis markers (cleaved caspase 3 and BCL2 expression) between vehicle and calcitriol treated tumorgrafts was detected. Additionally, no difference between groups was noticed for the expression of CDKN1A mRNA. In summary, in this small study, calcitriol antitumoral effects were not observed in tumorgrafts, established from highly proliferative breast cancer samples. This preclinical model in conjunction with pharmacokinetic calcitriol determinations might contribute to clarify the hormone effects in breast cancer.

Citation Format: Maria Lucia Hirata Katayama, Victor Celso N. Fonseca-Filho, Eduardo Carneiro Lyra, Durvanei Augusto Maria, Ricardo Alves Basso, Suely Nonogaki, Juliana Mariotti Guerra, Simone Maistro, João Carlos Sampaio Goes, Maria Aparecida A. Koike Folgueira. Calcitriol effects on breast cancer tumorgrafts. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3115. doi:10.1158/1538-7445.AM2014-3115
Abstract 312: Autophagy mediates HIF2α degradation and suppresses renal tumorigenesis
Authors: Liu Xian-De , Yao Jun , Tripathi Durga N. , Ding Zhiyong , Xu Yi , Sun Mianen , Zhang Jiangwei , Bai Shanshan , German Peter , Hoang Anh , Zhou Lijun , Zhang Xuesong , Conti Claudio J. , Efstathiou Eleni , Eissa Tony N. , Mills Gordon B. , Walker Cheryl L. , Jonasch Eric
Published: May 2015

Autophagy is a conserved process involved in lysosomal degradation of protein aggregates and damaged organelles. The role of autophagy in cancer is a topic of intense debate, and the underlying mechanism is still not clear. Here we reported that the hypoxia inducible factor 2α (HIF2α), an oncogenic transcription factor implicated in renal tumorigenesis, was in part constitutively degraded by autophagy. HIF2α interacts with autophagy protein LC3, autophagy receptor p62, and lysosome protein LAMP1. Inhibition of autophagy results in HIF2α accumulation, while induction of autophagy promotes HIF2α degradation. The E3 ubiquitin ligase activity of the von Hippel-Lindau (VHL) is required for autophagic degradation of HIF2α. There is a complementary interaction between the proteasome and autophagy in HIF2α degradation. Autophagy inactivation in ATG5 knockout cells redirects HIF2α to proteasomal degradation, while proteasome inhibition induced autophagy and favored the HIF2α-p62 interaction. Importantly, we reported that clear cell renal cell carcinoma (ccRCC) was frequently associated with mono-allelic loss and/or mutation of autophagy related gene ATG7, and low expression level of autophagy genes correlated with ccRCC progression. This study sheds light on the anticancer role for autophagy in ccRCC tumorigenesis, and reveals constitutive autophagic degradation of HIF2α as a novel tumor suppression mechanism.

Citation Format: Xian-De Liu, Jun Yao, Durga N. Tripathi, Zhiyong Ding, Yi Xu, Mianen Sun, Jiangwei Zhang, Shanshan Bai, Peter German, Anh Hoang, Lijun Zhou, Xuesong Zhang, Claudio J. Conti, Eleni Efstathiou, Tony N. Eissa, Gordon B. Mills, Cheryl L. Walker, Eric Jonasch. Autophagy mediates HIF2α degradation and suppresses renal tumorigenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 312. doi:10.1158/1538-7445.AM2014-312
Abstract 3122: Combination effect of metformin with gemcitabine for gemcitabine-resistant pancreatic adenocarcinoma
Authors: Suzuki Keiichi , Takeuchi Osamu , Osaku Masayoshi , Yamada Yoshinori
Published: May 2015

Metformin (MET) is the first-line treatment for type-2 diabetes mellitus. Several epidemiological studies have revealed the anti-cancer effects of metformin, including against pancreatic ductal carcinoma (PDC). Gemcitabine (GEM) has become the standard chemotherapy for PDC but tolerance to GEM is an important issue.

We evaluated the anti-tumor effects of MET for GEM-resistant PDC in a xenograft mouse model. For this in vivo study, wild-type BxPC-3 was implanted into both flanks of 7-week-old female BALB/c nude mice. Mice were divided into four groups: (i) control (without any treatment); (ii) GEM-treated group; (iii) MET-treated group; and (iv) combined treatment group (GEM+MET). Mice were fed for 4 weeks. Estimated tumor volumes and body weights were measured each week. Treatments were initiated 2 weeks after implantation. MET (600 mg/kg) was administrated orally every day. GEM (100 mg/kg) was given by intraperitoneal injection every week.

The final tumor volumes (in g) were: control, 0.59±0.05; GEM, 0.32±0.07; MET, 0.42±0.08; GEM+MET, 0.23±0.06. The two groups treated with GEM had significantly reduced tumors compared with control, but there were no differences between control and MET groups. The anti-tumor effect of MET for BxG30 (the cell line for GEM-resistant PDC) was evaluated using the method described above. The final tumor volumes were: control, 0.26±0.05; GEM, 0.21±0.05; MET, 0.15±0.06; GEM+MET, 0.11±0.03. Tumor volumes were decreased significantly in GEM+MET groups compared with control. The treated control ratio (T/C%) was calculated: GEM, 80.2%; MET, 54.0%; GEM+MET, 47.2%. The anti-tumor effect of GEM for BxG30 was limited. The MET group showed satisfactory anti-tumor effects, but T/C% was <50% only in the GEM+MET group. This result revealed that combination therapy had excellent anti-tumor effects even for GEM-resistant PDC. The IC50 was 20.25 mM for GEM and 0.004 µg/mL for MET upon single-drug use.

Our results showed that MET has a partial anti-tumor effect for PDC. Combination therapy has an excellent effect not only for wild-type PDC but also for GEM-resistant PDC. These data suggest that MET could be used to treat PDC.

Citation Format: Keiichi Suzuki, Osamu Takeuchi, Masayoshi Osaku, Yoshinori Yamada. Combination effect of metformin with gemcitabine for gemcitabine-resistant pancreatic adenocarcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3122. doi:10.1158/1538-7445.AM2014-3122
Abstract 3124: Xenograft models for development of new drugs targeting fibroblast growth factor receptor (FGFR)
Authors: Zhang Lan , Zhang Juan , Shi Qian
Published: May 2015

The fibroblast growth factor receptors (FGFR) are tyrosine kinases that are present in many types of endothelial and tumor cells. There are 4 different fibroblast growth factor receptors (FGFR) in the cell: FGFR1, FGFR2, FGFR3, and FGFR4. The activation of FGFR pathway plays an important role in tumor angiogenesis, tumor growth, survival, as well as migration. Mutation or amplification/overexpresion of FGFR has been found in several types of tumors, including breast cancer, bladder cancer, gastric cancer, prostate cancer, colon cancer, multiple myeloma, and non-small cell lung carcinoma. Therefore, targeting FGFRs represents an attractive strategy for development of new cancer therapeutics. However, the lack of suitable models hinders the progress of research on FGFR inhibitors. Here we successfully validated subcutaneous models in several cancer types, in which FGFR is mutant or amplified/overexpressed, such as breast cancer MDA-MB-134 (FGFR1 amplification), gastric cancer SNU-16 (FGFR2 amplification), endometrial cancer AN3CA (FGFR2 N549K/K310R mutation), bladder cancer RT-4 (FGFR3 overexpression) and RT-112 (FGFR3 overexpression), lung cancer NCI-H1581 (FGFR1 amplification), as well as human derived gastric cancer GA114 (FGFR2 amplification). In conclusion, the validated cell line derived (CDX) and patient derived xenograft models (PDX) in several cancer types provide valuable platforms for the development of new compounds targeting FGFRs.

Citation Format: Lan Zhang, Juan Zhang, Qian Shi. Xenograft models for development of new drugs targeting fibroblast growth factor receptor (FGFR). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3124. doi:10.1158/1538-7445.AM2014-3124
Abstract 3126: Renca RCC syngeneic model to evaluate efficacy of novel antisense oligonucleotides targeting TGF-β isoforms
Authors: Korhonen Hanna , Redon Julie-Orlane , France Damien , Serin Guillaume , Bichat Francis , Jaschinski Frank , Wosikowski Katja , Janicot Michel
Published: May 2015

Transforming growth factor beta (TGF-β represents a family of cytokines, which function as the primary mediators for TGF-β signaling via TGF-β receptor type II (TβIIR) and both non-canonical and canonical downstream signaling pathways. TGF-β is associated with a wide range of biological processes in oncology, including tumor cell invasion, migration, angiogenesis, immunosuppression, as well as regulation of tumor stem cell properties. Hence, optimal preclinical evaluation of efficacy of TGF-β antagonists is challenging. Isarna Therapeutics has designed and developed selective and potent LNA-modified antisense oligonucleotides targeting the various TGF-β isoforms. In order to adequately evaluate selected preclinical development candidates, Oncodesign has developed customized experimental Renca tumor models in syngeneic and/or immunodeficient mice. The Renca cell line was established from a murine transplantable renal adenocarcinoma of spontaneous origin, and has been used under various experimental conditions: (1) subcutaneous tumor model by inoculating cells into the flanks of the animals; (2) the pulmonary metastatic tumor model by an intravenous injection of cells into the tail vein; and (3) the orthotopic tumor model by injecting cells into the renal subcapsule (and subsequent pulmonary metastasis). Outcome of this development program and preliminary results for selected TGF-β antisense oligonucleotides will be presented and discussed.

Citation Format: Hanna Korhonen, Julie-Orlane Redon, Damien France, Guillaume Serin, Francis Bichat, Frank Jaschinski, Katja Wosikowski, Michel Janicot. Renca RCC syngeneic model to evaluate efficacy of novel antisense oligonucleotides targeting TGF-β isoforms. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3126. doi:10.1158/1538-7445.AM2014-3126
Abstract 3127: The effect of Guizhi Fuling Wan (GFW) on bladder tumor growth in a mouse model
Authors: Lu Chi-Chen , Tseng Ling-Huei , Chen Syue-Yi , Wu Jiann-Der , Wu Shu-Fen , Chan Michael W.Y. , Leu Yu-Wei , Hsu Cheng-Da
Published: May 2015

Bladder cancer is the second most common malignancy of the genitourinary tumors in the world and tenth most common cancer in Taiwan. The most common type of bladder cancer is urothelial carcinoma (UC, formerly known as transitional cell carcinoma, TCC). The incidence rate is particularly high in southwestern coast of Taiwan. Treatment for superficial non-invasive bladder carcinoma is currently using intravesical chemotherapy with mitomycin C and BCG after transurethral resection of bladder tumor (TUR-BT). However, this therapy usually causes adverse effects including urinary frequency, urinary urgency, hematuria and tuberculosis infection. Therefore, we intend to find a complementary and alternative medicine for bladder cancer treatment which can bring a good result with less side effects. In our previous study, we found that a traditional Chinese Medicine (TCM), Guizhi Fuling Wan (GFW) had an effect on growth inhibition of human bladder cancer cell lines as same as mitomycin-C, doxorubicin and cisplatin, but had mild toxicicity on normal human bladder epithelial cells. In this study we investigated whether GFW could be used as an intravesical chemotherapy agent in our orthotopic bladder cancer mouse model and compared its adverse effect with mitomycin-C and BCG. A mouse bladder cancer cell line, MB49, was selected and evaluated its response to GFW treatment. Cell Counting Kit-8 (CCK-8) assay was employed to analyze cell viability and flow cytometry was used to determine cell cycle and apoptosis. MB49 cells were implanted into female C57BL/6 mice to generate our orthotopic bladder tumor model which was confirmed under a microscope in histology by hematoxylin and eosin staining. Our data showed that GFW was a potent inhibitor of the proliferation of mouse bladder cancer cells in vitro as well as inhibited the tumor growth on MB49 bladder cancer orthotopic model. In addition, bladder hemorrhage which was observed in bladder cancer mice had completely disappeared 17 days after the intravesical GFW-treatment. These results support GFW as a strong candidate for intravesicle chemotherapy against bladder cancer.

Citation Format: Chi-Chen Lu, Ling-Huei Tseng, Syue-Yi Chen, Jiann-Der Wu, Shu-Fen Wu, Michael W.Y. Chan, Yu-Wei Leu, Cheng-Da Hsu. The effect of Guizhi Fuling Wan (GFW) on bladder tumor growth in a mouse model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3127. doi:10.1158/1538-7445.AM2014-3127
Abstract 3130: Zoledronic acid inhibits proliferation and metastasis of human pancreatic cancer in the patient-derived orthotopic xenograft (PDOX) model by targeting tumor-educated macrophages
Authors: Hiroshima Yukihiko , Hassenein Mohamed K. , Menen Rhiana , Katz Matthew H.g. , Fleming Jason B. , Sato Sho , Murakami Takashi , Yamamoto Mako , Uehara Fuminari , Miwa Shinji , Yano Shuya , Momiyama Masashi , Maawy Ali , Chishima Takashi , Tanaka Kuniya , Bouvet Michael , Endo Itaru , Hoffman Robert M.
Published: May 2015

Macrophages harvested from the peritoneal cavity of nude mice with subcutaneous human pancreatic tumors were defined as “tumor-educated macrophages” (EMϕ) and macrophages harvested from mice without tumors were defined as “naïve macrophages” (NMϕ). EMϕ promote tumor growth and metastasis. The aim of the present study was to determine the efficacy of Zoledronic acid (ZA) on EMϕ in a pancreatic cancer patient derived orthotopic xenograft (PDOX) nude mouse model. In this study, EMϕ and NMϕ were compared for their ability to enhance tumor progression. We initially demonstrated that the cancer cells exposed to the conditioned medium harvested from EMϕ culture significantly increased proliferation (p = 0.016) and had more migration stimulation capability (p < 0.001) compared to cultured cancer cells treated with the conditioned medium from NMϕ. Next, we examined the efficacy of ZA on EMϕ, and NMϕ and found that ZA had the ability to kill both EMϕ and NMϕ in vitro. We then demonstrated that EMϕ promoted tumor growth and metastasis in an orthotopic mouse model of a human pancreatic cancer cell line, ZA reduced the tumor growth (p = 0.006) and metastasis (p = 0.025) promoted by EMϕ. Finally, we examined the efficacy of ZA for pancreatic cancer in the PDOX model, and found that the combination of gemcitabine (GEM) and ZA reduced tumor weight (p = 0.016) and tumor growth (p = 0.005) compared to GEM alone. ZA alone reduced metastasis (p = 0.009). These results suggest that ZA inhibits the proliferation and the metastasis of human pancreatic cancer by targeting EMϕ.

Citation Format: Yukihiko Hiroshima, Mohamed K. Hassenein, Rhiana Menen, Matthew H.g. Katz, Jason B. Fleming, Sho Sato, Takashi Murakami, Mako Yamamoto, Fuminari Uehara, Shinji Miwa, Shuya Yano, Masashi Momiyama, Ali Maawy, Takashi Chishima, Kuniya Tanaka, Michael Bouvet, Itaru Endo, Robert M. Hoffman. Zoledronic acid inhibits proliferation and metastasis of human pancreatic cancer in the patient-derived orthotopic xenograft (PDOX) model by targeting tumor-educated macrophages. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3130. doi:10.1158/1538-7445.AM2014-3130
Abstract 3131: Recurrence of cervical cancer following PR agonist withdrawal in mice
Authors: Mehta Fabiola , Ojha Salil , Chung Sang-Hyuk
Published: May 2015

The majority of human cervical cancers are associated with the high-risk human papillomaviruses (HPVs). Their tumorigenic potential stems mainly from viral oncogenes E6 and E7, which are best known to inactivate p53 and pRb tumor suppressor, respectively. Epidemiological studies, however, have suggested that persistent HPV infections are not sufficient and other cofactors are necessary for the progression of the disease. Prior studies using HPV transgenic mice expressing E6 and E7 under the control of K14 promoter (K14E6/K14E7) have demonstrated the requirement of estrogen and estrogen receptor alpha (ERα) for the development of HPV-induced cervical cancer. It was also shown that inhibition of ERα is effective in both treating and preventing cervical cancer in the HPV transgenic mouse model. Another female sex hormone, progesterone, functions through the progesterone receptor (PR), which is a direct transcriptional target of ERα. PR and ERα are transcription factors, members of the nuclear receptor superfamily. We recently demonstrated that a PR agonist, medroxyprogesterone acetate (MPA), is efficient in regressing cervical cancer and cervical intraepithelial neoplasia (CIN) in vivo. This result suggests that PR has an anti-tumorigenic activity in the context of ERα-dependent cervical cancer. In the present study, we investigated whether cervical cancer recurs after stopping MPA treatment using the same mouse model. Consistent with our previous publication, all K14E6/K14E7 double transgenic mice treated with estrogen for 6 months had cervical cancer, which regressed when treated with MPA for 2 months. When the mice were re-treated for 2 months with estrogen after MPA treatment, cervical cancer was observed in all mice. Interestingly, the cancer was also present in all mice that were left untreated for 2 months after MPA treatment. These results suggest that cervical cancer recurs if MPA treatment is ceased. The mice bearing recurring cancer were treated with MPA again for 2 months. It was interesting that the cancer remained in 6 of 7 mice, although cancers deemed smaller than those recurred. Further analyses of these cancers are warranted. Taken together, we conclude that cervical cancer recurred once MPA was withdrawn and recurring cancers partially responded to MPA. It implies that MPA treatment regimen used in the study did not eliminate all cancerous cells. Our results suggest that, if translatable to women, continuation of MPA treatment will be warranted to prevent the recurrence of cervical cancer.

Citation Format: Fabiola Mehta, Salil Ojha, Sang-Hyuk Chung. Recurrence of cervical cancer following PR agonist withdrawal in mice. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3131. doi:10.1158/1538-7445.AM2014-3131
Abstract 3136: X-MAN™ isogenic DualXenoTM models with KRAS mutation predicts the effect of anti-EGFR agents
Authors: Sun Yanmei , Zhang Songling , Li Nan , Astley Holly , Foster Rebecca , Schofield Christine , Torrance Chris Chris , Ning Jinying , Shi Qian
Published: May 2015

We have previously reported using cell lines generated with Horizon Discovery's rAAV-based GENESIS™ gene editing platform to establish a comprehensive list of isogenic cancer models for in vivo compound screening, with mutations in a wide variety of genes including KRAS, PIK3CA, PTEN, IDH1 and IDH2, and p53. These isogenic tumor models comprise pairs of cell lines which share the same genetic background, differing only by the mutation of interest, and therefore allow definitive studies of specific genetic variances to be performed.

In the current study we developed a DualXenoTM method where isogenic pairs of a colorectal cell line, one of each pair with a KRAS mutation, were inoculated simultaneously in the two flanks of the same mouse. The tumors were then treated with EGFR targeted therapeutics to address the question of resistant phenotypes elicited by different KRAS mutations. This design allows direct comparison of wild type and mutant isogenic pairs for treatment responses that are associated with the defined genetic variations. Our results demonstrated that tumors harboring the G12V mutation were resistant to both Cetuximab and Erlotinib treatment, while tumors harboring the G13D mutation remained sensitive to both agents. This is in consistent with clinical findings (De Roock, et al. JAMA 2010, 304(16), pp 1812), and our own findings with PDX mouse clinical trials in colon cancer, suggesting that the KRAS G13D mutation may establish a different signaling network to other KRAS mutations, and that colon cancer patients with the mutation should not be excluded from the EGFR targeted therapies.

Citation Format: Yanmei Sun, Songling Zhang, Nan Li, Holly Astley, Rebecca Foster, Christine Schofield, Chris Chris Torrance, Jinying Ning, Qian Shi. X-MAN™ isogenic DualXenoTM models with KRAS mutation predicts the effect of anti-EGFR agents. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3136. doi:10.1158/1538-7445.AM2014-3136