bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3041: GPR56 promotes the adhesion of glioma stem-like cells to the perivascular niche and regulates cell fate
Authors: Moreno Marta , Giera Stefanie , Piao Xianhua , Iglesia Nuria de la
Published: May 2015

INTRODUCTION

Gliobastomas (GBM) are the most aggressive and frequent human malignancies in the central nervous system. A small population of cells within the bulk of these tumors displays properties of normal neural stem cells (NSC). Accordingly, these cells are referred to as glioma stem-like initiating cells (GIC). Thus, deregulation of mechanisms that control NSC biology might contribute to the pathogenesis of GBM. NSC and GIC are localized to the perivascular space where they reside in close apposition to blood vessels (BV), which promote self-renewal and keep these cells in an undifferentiated state. GPR56 is an adhesion G-protein coupled receptor expressed in neural progenitors in the embryonic and adult brain and plays an indispensable role in cortical development. Knockout mice studies show that GPR56 mediates the attachment of radial glial endfeet to pial basement membrane (BM). Some of the components of the pial BM are also present in the extracellular matrix surrounding BV, such as laminin and collagen. Therefore, we hypothesized that GPR56 might mediate NSC/GIC localization to the perivascular niche thus contributing to their stem-related properties.

EXPERIMENTAL PROCEDURES

Here, we have compared the expression of GPR56 in neural progenitors versus more differentiated progeny. In addition, we have assessed the role of GPR56 in the neural stem-like properties of GIC, including their differentiation into more mesenchymal cell types such as endothelial cells or pericytes, by gain- and loss-of-function studies.

RESULTS

We have found that GPR56 is highly expressed in NSC and its expression is downregulated during differentiation. We have also observed that GPR56 is expressed in cells that reside in the perivascular niche. GPR56-knockdown GIC display impaired adhesion to endothelial cells (EC), whereas GPR56 overexpression increases their adhesion to EC. Furthermore, loss of GPR56 increases the ability of GIC to express endothelial/pericyte markers when co-cultured with EC and increases the CD44+ population, suggesting that GPR56 inhibits the acquisition of a mesenchymal-like phenotype.

CONCLUSIONS

Taken together, our findings indicate that GPR56 mediates the adhesion of GIC to EC. Moreover, GPR56 inhibits the acquisition of mesenchymal features. Therefore, we suggest that GPR56 might mediate the attachment of GIC to the perivascular niche while preserving their neural stem-like properties by preventing the acquisition of a mesenchymal-like phenotype.

Citation Format: Marta Moreno, Stefanie Giera, Xianhua Piao, Nuria de la Iglesia. GPR56 promotes the adhesion of glioma stem-like cells to the perivascular niche and regulates cell fate. [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 3041. doi:10.1158/1538-7445.AM2014-3041
Abstract 3067: PIK3CA hotspot mutations are present at a relatively high frequency in CTCs of operable and metastatic breast cancer patients
Authors: Markou Athina N. , Farkona Sofia , Schiza Christina , Eftathiou Antonia , Malamos Nikolaos , Georgoulias Vassilis , Lianidou Evi
Published: May 2015

Introduction: Molecular characterization of CTC is crucial for the investigation of molecular targeted therapies. PIK3CA somatic mutations play a crucial role in response to molecular targeted therapies. We detected PIK3CA mutations at a high frequency in CTC after developing and validating an ultra-sensitive methodology, based on a combination of allele-specific, asymmetric rapid PCR and melting analysis.

Patient and Methods: After optimizing and validating our assay in terms of sensitivity, specificity and robustness, we detected PIK3CA hotspot mutations in EpCAM positive CTCs from 57 metastatic and 118 early breast cancer patients, 26 healthy individuals and 76 corresponding primary tumors. Rapid Real-PCR and melting were performed in triplicate for all samples in the LightScanner32 (Idaho USA), in the presence of LC-Green Plus saturating dye.

Results: The assay could detect 0.05% of mutated dsDNA in the presence of 99.95% wtDNA for both exons and was highly specific (0/26 healthy). We identified PIK3CA mutations in EpCAM positive CTC in 20/57(35.1%) metastatic and in 23/118(19.5%) operable breast cancer patients. In corresponding primary tumors, 45/76(59.2%) samples were positive. Patients with verified metastasis carrying PIK3CA mutations on CTC had significant shorter OS than those without.

Conclusions: We report for the first time that PIK3CA hotspot mutations are present at a relatively high frequency in CTC both in metastatic and operable breast cancer. The presence of PIK3CA mutations in CTC is associated with worse survival in patients with verified metastasis. Evaluation of PIK3CA mutational status on CTCs is a strategy with potential clinical application.

Citation Format: Athina N. Markou, Sofia Farkona, Christina Schiza, Antonia Eftathiou, Nikolaos Malamos, Vassilis Georgoulias, Evi Lianidou. PIK3CA hotspot mutations are present at a relatively high frequency in CTCs of operable and metastatic breast cancer patients. [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 3067. doi:10.1158/1538-7445.AM2014-3067
Abstract 307: The use of biospecimens in cancer population science research
Authors: Carrick Danielle M. , Mette Eliza , Hoyle Brittany , Rogers Scott D. , Gillanders Elizabeth M. , Schully Sheri D. , Mechanic Leah E.
Published: May 2015

Background: Over the past two decades, researchers have increasingly used human biospecimens to help evaluate hypotheses related to disease risk, outcomes and treatment options. We conducted an analysis of the population science cancer research grants funded by NCI in order to gain a more comprehensive understanding of the biospecimens involved in those studies. Recognizing that there are many pre-existing collections of biospecimens, we investigated the cost and time efficiencies observed with studies involving the use of existing biospecimens versus collecting new specimens. Methods: Data available for 1,018 extramural, peer-reviewed grants (active as of July 2012) supported by the Division of Cancer Control and Population Sciences, (NCI Division that supports cancer control and population science extramural research grants) were analyzed. Results: 455 of the grants were determined to involve biospecimens. The most common specimen types included were DNA (66% of grants involved DNA), whole blood (51%), serum or plasma (40%), and tissue (39%). Grants that involved the use of existing biospecimens resulted in greater cost (serum/plasma studies were 4.2 times less expensive) and time efficiencies (1.4 times more publications per year resulted) than grants that collected new biospecimens. Conclusions: Clearly, there is an opportunity for NCI to promote future sharing. We are currently working to better catalogue our funded resources and make data available to the extramural community. Further work is being done to investigate possible trends based on year of grant award.

Citation Format: Danielle M. Carrick, Eliza Mette, Brittany Hoyle, Scott D. Rogers, Elizabeth M. Gillanders, Sheri D. Schully, Leah E. Mechanic. The use of biospecimens in cancer population science research. [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 307. doi:10.1158/1538-7445.AM2014-307
Abstract 3071: Size-based isolation of circulating tumor cells (CTCs) in mouse tumor models
Authors: Kolostova Katarina , Hoffman Robert M. , Maawy Ali , Zhang Yong , Bobek Vladimir
Published: May 2015

We report here isolation and cultivation of circulating tumor cells (CTCs) in five human epithelial cancers (lung, prostate, colon, pancreatic and breast cancer) implanted in nude mice. All of the cancer cells were labeled with green fluorescent protein (GFP) for detection. CTCs were isolated with an antigen-independent size-based separation device (MetaCell®, Metacell s.r.o., Ostrava, Czech Republic). We were able to capture on average more than 2 cells for each of above cancer types whereby the tumor has been implanted atleast 2 weeks before blood withdrawal. The number of CTC is dependent on the stage of tumor in animals. There was a very high heterogeneity of the CTCs with regard to cell size and cell phenotype including epithelial- and mesenchymal-like type cells in vitro. The development of a facile in vitro isolation and imaging technology for CTCs will enable deeper understanding of CTC biology.

Citation Format: Katarina Kolostova, Robert M. Hoffman, Ali Maawy, Yong Zhang, Vladimir Bobek. Size-based isolation of circulating tumor cells (CTCs) in mouse tumor models. [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 3071. doi:10.1158/1538-7445.AM2014-3071
Abstract 3072: High-recovery multiplex analysis of circulating tumor cells by density-based enrichment, automated platform immunofluorescence staining, and digital microscopy
Authors: Campton Daniel , Ramirez Arturo , Nordberg Joshua , Blau Anthony , Stilwell Jackie , Kaldjian Eric
Published: May 2015

Background: Analysis of circulating tumor cells (CTC) is an intense area of diagnostic research that is rapidly evolving from prognostic to therapeutic applications. We applied three sequential technologies - density-based enrichment, automated immunofluorescence staining, and digital microscopy with image analysis to the investigation of recovery rates from blood containing spiked-in tumor cells as a model of CTC.

Materials and Methods: Cultured MCF7, PC3, A549 and LNCaP cells were counted using Vitrotube visualization. ∼100 cells per line were spiked into normal blood samples in two experiments. Density-based enrichment was performed using the AccuCyte tube, float and collector system in a single-tube, two-spin process. Collected cells were processed with an adherence solution and applied to charged microscopic slides with a simple spreading device. After drying, slides were placed onto the Ventana Discovery Ultra automated platform and fluorescently stained with antibodies to cytokeratin, CD45, and EpCAM or EGFR, as well as a nuclear dye. Slides were scanned on a CyteFinder digital microscope and candidate CTCs were identified using CyteMapper image analysis software. CTCs were verified by a reviewer by appropriate morphology and expression of both epithelial and nuclear stains without CD45 expression.

Results: In a total of seven samples the recovery of spiked-in CTCs ranged from 88 to 98% and averaged 91%. This method was successfully applied to samples of blood from patients with triple-negative breast cancer.

Conclusions: Density-based enrichment combined with automated immunofluorescence staining, digital microscopy and confirmation of candidate cells by a trained reviewer, produces exceptional recovery of CTCs and can be used to assess clinical samples.

Recovery of spiked-in CTCsCell line - exptSpiked-in cellsCTCs countedRecoveryLNCaP - 112711691%LNCaP - 212310989%A549 - 1868498%A549 - 212411593%MCF7 - 11099688%MCF7 - 21049288%PC3 - 2888192%

Citation Format: Daniel Campton, Arturo Ramirez, Joshua Nordberg, Anthony Blau, Jackie Stilwell, Eric Kaldjian. High-recovery multiplex analysis of circulating tumor cells by density-based enrichment, automated platform immunofluorescence staining, and digital microscopy. [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 3072. doi:10.1158/1538-7445.AM2014-3072
Abstract 3073: Detection and isolation of circulating tumor cells in ovarian cancer: Principles and methods
Authors: Lee Shin-Wha , Lee Ha-Young , Mo Jin-Young , Kim Byung Chul , Kim Eun Hye , Kim Yong-Man
Published: May 2015

Objective:

The quantitative and qualitative analysis of circulating tumor cells (CTCs) aids in diagnosis of disease, prognosis, disease recurrence, and therapeutic response in several cancers. However, detection and isolation of CTCs from the blood circulation is a difficult task because of their scarcity and the lack of reliable markers to identify these cells. Especially, in epithelial ovarian cancer, there is a little reported data for CTCs and their CTCs has been defined to the EpCAM+ cells, even though EpCAM+ cells are just a few in ovarian cancer. The aim of this study is to investigate the effective principles and methods to detect and isolate CTCs through novel markers in ovarian cancer.

Methods:

CTC analysis was performed in the spiking test to find cancer cells in the human serum mixed with WBCs. Four cell lines including OVCAR3, SKOV3, SNU8 and SNU251 were used and primary cancer cells from 4 patients were analyzed. Cancer cells were isolated on the basis of cell size by filtration through CytoGen capture device, followed by identification according to validated immunocytochemistry based on the expression of DAPI, CD45, EpCAM, CK, CA-125 and HE4.

Results:

We obtained cancer cells more than 90% based on the cell size by filtration. Immunocytochemistry confirmed the epithelial origin through the expression of CK and EpCAM in cancer cell lines. The rate of expression was 35-99 % for CK and 1-99 % for EpCAM. A small number of cancer cell lines expressed CD45 that is the marker of WBC. The expression of CA-125 and HE4 showed their ovarian origin. The rate of expression was 7-98 % for CA-125 and 40-99 % for HE4 in cancer cell lines. Primary cancer cells were detected most effectively as the method of combination with 4 markers, including DAPI, CD45, EpCAM and HE4.

Conclusion:

Our findings provide the first evidence for effective methods to detect and isolate CTCs in ovarian cancer. Combination of DAPI, CD45, EpCAM and HE4 could be a useful marker to support CTCs research in epithelial ovarian cancer.

Citation Format: Shin-Wha Lee, Ha-Young Lee, Jin-Young Mo, Byung Chul Kim, Eun Hye Kim, Yong-Man Kim. Detection and isolation of circulating tumor cells in ovarian cancer: Principles and methods. [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 3073. doi:10.1158/1538-7445.AM2014-3073
Abstract 3074: Detection and enumeration of circulating tumor cells using imaging flow cytometery
Authors: Vaidyanathan Shobana , Weldon Don , Basiji David , Morrissey Philip
Published: May 2015

Circulating tumor cells (CTCs), released into the bloodstream from primary and metastatic cancers, are valuable tools for understanding tumor biology. However, since they are rare, their detection is hampered by low efficiency and a lack of standardization in current technologies. To overcome these problems, we used Imaging Flow Cytometery along with fluorescent RNA detection probes to collect imagery from large number of cells to assess the number of CTCs. In this study, we spiked peripheral blood mononucleated cells (PBMCs) with SKBR-3 human breast cancer cells and added probes for relevant RNA targets such as EPCAM and Her-2. Taking advantage of the probes’ ability to detect RNA in live cells, combined with the capacity to acquire multi-spectral images of large numbers of cells, we demonstrate image based parameters that can be used to assess the frequency of CTCs in an objective and statistically significant manner.

Citation Format: Shobana Vaidyanathan, Don Weldon, David Basiji, Philip Morrissey. Detection and enumeration of circulating tumor cells using imaging flow cytometery. [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 3074. doi:10.1158/1538-7445.AM2014-3074
Abstract 3086: The genetic landscape of the childhood liver cancer hepatoblastoma
Authors: Kappler Roland , Eichenmüller Melanie , Trippel Franziska , Strom Tim M. , Schweinitz Dietrich von
Published: May 2015

Hepatoblastoma (HB) is the most common malignant liver tumor in children with a histology that resembles various stages of the developing liver. To explore the genetic origin of this cancer, we performed whole-exome sequencing of 8 tumors and matched germline tissue. In average, we detected only three sequence variants per tumor, which is the lowest mutation rate ever reported for any cancer. Mutations of ß-catenin (CTNNB1) displayed the only recurrent event occurring in all tumors, thereby underscoring the significance of activated Wnt signaling in the genesis of HB. Gene ontology analysis revealed a predominance of candidate genes involved in the regulation of gene expression and transcription, including the nuclear factor (erythroid-derived 2)-like 2 (NFE2L2). Targeted resequencing of a cohort of 50 HB cases and cell lines revealed NFE2L2 mutations in 10% of all tumors. Interestingly, all mutations were located in or adjacent to the DLG and ETGE motifs of the NFE2L2 protein that are known to be recognized from the KEAP1/CUL3 complex for proteasomal degradation. In line with this, cells transfected with mutant NFE2L2 were insensitive to KEAP1-mediated downregulation of NFE2L2 signaling. Clinically, overexpression of the NFE2L2 target gene NQO1 in tumor tissues was significantly associated with high tumor stage, metastasis, and poor outcome. These data suggest that activation of NFE2L2-KEAP1 signaling through mutations in NFE2L2 is a prominent characteristic in pediatric liver tumors, thereby advocating inhibition of aberrant NFE2L2 activity as a therapeutic option, especially in high-risk patients with advanced disease.

Citation Format: Roland Kappler, Melanie Eichenmüller, Franziska Trippel, Tim M. Strom, Dietrich von Schweinitz. The genetic landscape of the childhood liver cancer hepatoblastoma. [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 3086. doi:10.1158/1538-7445.AM2014-3086
Abstract 31: Phosphatidylserine-specific phospholipase A1 (PSPLA1) expression in colorectal cancer correlates with tumor invasion and hematogenous metastasis
Authors: Iida Yuuki , Sunami Eiji , Ishihara Soichiro , Yamaguchi Hironori , Tsuno Nelson H. , Kitayama Joji , Watanabe Toshiaki
Published: May 2015

Background: Although the physiological functions of various phospholipases, such as lysophospholipase D (Autotaxin), has been extensively investigated, those of Phosphatidylserine-Specific Phospholipase A1 (PSPLA1), a kind of phospholipase involved in allergic reactions, has not been fully elucidated yet. Here, we evaluated the role of PSPLA1 in oncogenesis and tumor metastasis of colorectal cancer.

Materials and Methods: Surgical specimens of 85 consecutive patients with colorectal cancer who received surgery in our department between January 2005 and December 2005 were immunostained with a monoclonal antibody to PSPLA1. The correlation between PSPLA1 expression and the clinico-pathological variables was analyzed by univariate and multivariate analyses. The impact of PSPLA1 expression on the prognosis of the patients was assessed, in comparison with the other clinico-pathological variables.

Results: PSPLA1 expression significantly correlated with tumor size, tumor depth and hematogenous metastasis in univariate analysis, and tumor depth and hematogenous metastasis were independent variables in multivariate analysis. The disease-free survival (DFS) of patients with high-PSPLA1 expression was significantly shorter compared to those with low expression. The presence of hematogenous metastasis, however, was the only independent factor associated with poor DFS.

Conclusion: PSPLA1 expression in colorectal cancer was associated with tumor invasion and metastasis.

Citation Format: Yuuki Iida, Eiji Sunami, Soichiro Ishihara, Hironori Yamaguchi, Nelson H. Tsuno, Joji Kitayama, Toshiaki Watanabe. Phosphatidylserine-specific phospholipase A1 (PSPLA1) expression in colorectal cancer correlates with tumor invasion and hematogenous metastasis. [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 31. doi:10.1158/1538-7445.AM2014-31
Abstract 3109: ALK as a valid therapeutic target for the treatment of rhabdomyosarcoma
Authors: Wierdl Monika , Tsurkan Lyudmila , Tollemar Viktor , Chi Lying , Stewart Elizabeth , Dyer Michael A. , Potter Philip M.
Published: May 2015

Anaplastic Lymphoma Kinase (ALK) is a transmembrane receptor tyrosine kinase, a member of the insulin receptor family, with a tightly controlled expression pattern. Deregulated ALK expression has been linked to the development of several types of cancers. Typically, chromosomal translocations involving ALK generate constitutively expressed chimeric oncoproteins, such as NPM-ALK in anaplastic large cell lymphoma and EML4-ALK in non-small cell lung carcinoma. However, activating mutations in ALK, and increased copy number that result in aberrant gene expression, have also been observed in neuroblastoma and colorectal cancers. Based on the above observations, several small molecule ALK inhibitors have been developed and are in clinical trials for a variety of tumors expressing this kinase. This has resulted in the use of crizotinib for the treatment of EML4-ALK + lung cancers.

Recently several large scale immunohistochemistry and genome sequencing studies of pediatric rhabdomyosarcoma (RMS) revealed aberrant ALK expression. It was found that ALK mRNA expression was significantly different in alveolar (ARMS) and embryonic RMS (ERMS), the two major histological subtypes in children. Thus, ALK expression might play a role in tumorigenesis for this disease, and this kinase may represent a valid therapeutic target for the treatment of RMS. To answer this question we analyzed ALK expression in a large panel of RMS cell lines, and pediatric tumor samples by quantitative PCR and immunoblotting. We also compared the sensitivity of these cells to a series of ALK inhibitors. Our results indicate that the majority of ARMS cell lines demonstrated significantly higher ALK mRNA expression as compared to ERMS, and to normal human myoblast control cells. This was confirmed by western analysis. Growth inhibition assays confirmed that all RMS lines were more sensitive to crizotinib and LDK-378 than control lines, consistent with the hypothesis that ALK is a driver of cell growth in these tumors.

Based on our in vitro analysis, we suggest that ALK inhibition may represent an effective therapeutic modality for the treatment of ALK+ RMS. Preclinical studies to confirm or disprove this hypothesis are currently underway.

Supported by St. Jude Children's Research Hospital and by the American Lebanese Syrian Associated Charities

Citation Format: Monika Wierdl, Lyudmila Tsurkan, Viktor Tollemar, Lying Chi, Elizabeth Stewart, Michael A. Dyer, Philip M. Potter. ALK as a valid therapeutic target for the treatment of 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 3109. doi:10.1158/1538-7445.AM2014-3109
Abstract 3110: Targeting the Wnt/PCP signaling through ROCK: A new neuroblastoma drug target
Authors: Dyberg Cecilia , Forsberg David , Papachristou Panos , Lannerholm-Palm Jessika , Haug Bjorn Helge , Sveinbjörnsson Baldur , Lagercrantz Hugo , Kogner Per , Johnsen John inge , Wickstrom Malin
Published: May 2015

Background: The non-canonical Wnt/planar cell polarity (PCP) pathway regulates cytoskeletal organization, migration and neuritogenesis. Signaling is characterized by activation of the GTPases Rho and Rac, and the downstream Rho-associated protein kinases (ROCKs). Neuroblastoma is a malignant embryonal tumor of the sympathetic nervous systems, often with poor prognosis. The need for novel therapeutic approaches is great. Genetic analyses have revealed mutations and aberrations in the regulators Rho/Rac in neuroblastoma. The aim of this study was to characterize the Wnt/PCP signaling and the effects of ROCK inhibition in neuroblastoma.

Methods: Cytotoxic activity of ROCK inhibitors was studied in cell viability assays. Morphology and invasion were studied with microscopy. The molecular mechanisms were characterized using cell- and molecular biology techniques. In vivo studies in mice were carried out to validate the therapeutic effects and toxicity.

Results: Several mediators in the pathway were differentially expressed in cell lines and patient samples. Using compounds blocking ROCK1 and ROCK2 activity revealed that the ROCK2 inhibitor HA-1077 effectively repressed proliferation and reduced cell viability in neuroblastoma. Additionally, HA-1077 inhibited migration and induced differentiation through initiating neural outgrowth. Furthermore, HA-1077 reduced the growth of established neuroblastoma xenografts in nude mice.

Conclusions: These results suggest that non-canonical Wnt signaling in general and ROCK in particular is a promising new therapeutic target for high-risk neuroblastoma.

Citation Format: Cecilia Dyberg, David Forsberg, Panos Papachristou, Jessika Lannerholm-Palm, Bjorn Helge Haug, Baldur Sveinbjörnsson, Hugo Lagercrantz, Per Kogner, John inge Johnsen, Malin Wickstrom. Targeting the Wnt/PCP signaling through ROCK: A new neuroblastoma drug target. [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 3110. doi:10.1158/1538-7445.AM2014-3110
Abstract 3118: Chemokine-targeted colorectal cancer: improved models of human primary tumor formation, liver metastasis and chemoresistance
Authors: Chen Huanhuan Joyce , Sun Jian , Hou Harry , Edelmann Winfried , Shen Xiling , Lipkin Steven M.
Published: May 2015

Current human colorectal cancer (CRC) orthotopic xenograft models require surgery and do not readily progress to form tumors in liver, the most common site for metastasis. We developed cell and patient-derived xenograft models that use chemokine-targeting to recapitulate the vast majority of recurrent human somatic CRC mutations as primary gastrointestinal (GI) tumors in mice without requiring surgery. Importantly, we incorporate early-stage mouse blastocyst microinjection techniques to extend this approach and demonstrate that it can also model primary human CRCs in immunoproficient mouse hosts. Next, we show that primary GI tumors can inducibly metastasize to liver. Finally, we use this system to demonstrate that human CRC liver metastases in vivo are more chemoresistant than paired sub-cutaneous xenografts. Overall, we anticipate that this resource can help improve our mechanistic understanding of human primary CRC growth, progression to liver metastasis and potentially provide a more physiological model than sub-cutaneous xenograft for pre-clinical drug discovery.

Citation Format: Huanhuan Joyce Chen, Jian Sun, Harry Hou, Winfried Edelmann, Xiling Shen, Steven M. Lipkin. Chemokine-targeted colorectal cancer: improved models of human primary tumor formation, liver metastasis and chemoresistance. [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 3118. doi:10.1158/1538-7445.AM2014-3118
Abstract 3135: Pre-treatment p-EGFR levels in tumors from a genetically engineered mouse model of BRAFV600E colorectal cancer predict response to combined BRAF/EGFR inhibition
Authors: Coffee Erin M. , Corcoran Ryan B. , Engelman Jeffrey A.
Published: May 2015

BRAFV600E colorectal cancer (CRC) patients do not exhibit responses to single-agent BRAF inhibition due to activation of EGFR. While combined BRAF and EGFR inhibition has shown some success in early clinical trials, there are still a subset of patients who do not respond to this combination. The purpose of this study is to identify which BRAFV600E tumors would be expected to respond to combined BRAF/EGFR inhibition based on the pre-treatment levels of pEGFR.

A genetically engineered mouse model (GEMM) for Apc-/- BrafV600E/+ p53-/- CRC was used for the study. Tumors were induced in the colons using adenovirus expressing Cre recombinase. Tumors were visualized by optical colonoscopy and pre-treatment tissue biopsy samples were obtained. BRAF mutant mice were randomized to a combination treatment with a BRAF inhibitor (Dabrafenib), combined with either an EGFR inhibitor (Erlotinib) or a MEK inhibitor (Trametinib). Tumor growth or regression was followed for 21 days by colonoscopy.

A retrospective analysis indicated that those tumors with high pre-treatment phosphorylated EGFR (p-EGFR) levels had significant responses to combined BRAF/EGFR therapy, while those tumors with little to no response to combined BRAF/EGFR therapy had low levels of p-EGFR prior to treatment. Furthermore, the majority of tumors that had a significant response to the combined BRAF/MEK therapy had the lowest levels of pre-treatment p-EGFR.

These pre-clinical data suggest that BRAFV600E CRC patients could benefit from being stratified by p-EGFR levels prior to treatment. Those patients with high p-EGFR levels would be expected to respond best to combined BRAF/EGFR treatment, while those patients with low p-EGFR levels might benefit from a different combination therapy, such as combined BRAF/MEK inhibition.

Citation Format: Erin M. Coffee, Ryan B. Corcoran, Jeffrey A. Engelman. Pre-treatment p-EGFR levels in tumors from a genetically engineered mouse model of BRAFV600E colorectal cancer predict response to combined BRAF/EGFR inhibition. [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 3135. doi:10.1158/1538-7445.AM2014-3135
Abstract 3144: PDZ-RhoGEF/β-arrestin-1 interaction mediates endothelin A receptor-induced RhoA activation and cell motility in ovarian tumor cells
Authors: Tocci Piera , Cianfrocca Roberta , Semprucci Elisa , Castro Valeriana Di , Bagnato Anna , Rosanò Laura
Published: May 2015

β-arrestins (β-arrs), consisting of β-arr1 and β-arr2, are involved in G-protein-coupled receptor signaling, and guide the receptor signals in chemotaxis and cytoskeletal reorganization. Among them, receptor A for endothelin-1 (ET-1), namely ETAR, is overexpressed in epithelial ovarian cancer (EOC) and promote metastatic spread. We have recently reported that β-arr1 regulates ETAR-mediated β-catenin signaling to promote EOC cell invasion. Here we evaluated the effects of ET-1 on polarized cell migration correlating with the regulation of Rho-GTPase activity and its subsequent organization of the actin structures supporting migration and the involvement of β-arr1 in these effects. We show that, in EOC cells cultured on collagen type I, ET-1 promotes actin-based dynamic protrusion resembling to pseudopodia, containing β-arr-1. Furthermore, in a β-arr1/ETAR-dependent manner, ET-1 activates RhoA GTPase and downstream the dephosphorylation/activation of cofilin, as shown by pull down assay and immunoblotting analysis. The inhibitory effect of the Rho pathway inhibitor I, Y-27632, demontrates that RhoA GTPase is involved in ET-1-induced cell migration. By screening members of a Rho-guanine nucleotide exchange factor (Rho-GEF) family involved in RhoA activation, we found that PDZ-RhoGEF mRNA levels are highly expressed in EOC cells. Moreover, PDZ-RhoGEF is responsible for ET-1-induced RhoA activation through interaction with β-arr1, that in turn, promotes PDZ-RhoGEF tyrosine phosphorylation and activation. Depletion of PDZ-RhoGEF expression significantly impaired ET-1-stimulated migration of EOC cells, demonstrating that PDZ-RhoGEF/β-arrestin-1 interaction mediates ETAR-driven ovarian tumor cell motility through regulation of RhoA. Analysis of EOC human tissues revealed that PDZ-RhoGEF is overexpressed in human EOC tissues. Collectively, our data establish a novel role for β-arr1 and PDZ-RhoGEF as regulator of ET-1-induced EOC cell migration, unraveling new components required for tumor progression. Supported by AIRC.

Citation Format: Piera Tocci, Roberta Cianfrocca, Elisa Semprucci, Valeriana Di Castro, Anna Bagnato, Laura Rosanò. PDZ-RhoGEF/β-arrestin-1 interaction mediates endothelin A receptor-induced RhoA activation and cell motility in ovarian 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 3144. doi:10.1158/1538-7445.AM2014-3144
Abstract 3165: Docosahexaenoic acid attenuates survival and progression in subtype-specific breast cancer by modifying Myc activity
Authors: Mouradian Michael , Bains Palvinder K. , Chattin Amy M. , Pardini Ronald S.
Published: May 2015

Overexpression of Myc in breast cancer is associated with poor prognosis and has a unique profile distribution among subtypes. Myc overexpression has been shown to be greatest in basal-like tumors, ∼50%, and least in luminal Her2-overexpressing tumors, ∼9%. Previous studies from our laboratory have identified a relationship between docosahexaenoic acid (DHA, C22:6 n-3) treatment and Myc activity and the effect seems to be specific to the breast cancer subtype and Myc overexpression profile, but is still unclear. In the current study, we investigated the effect of DHA on tumor growth, survival, and invasion in a high Myc expressing cell line, basal-like MDA-MB-231, and a low Myc expressing cell line, luminal Her2-overexpressing BT-474, in vitro. Basal expression analysis confirmed Myc overexpression levels in each cell line. While DHA treatment in the basal-like MDA-MB-231 cells significantly decreased Myc transcriptional activity and p-T58/S62-c-Myc phosphorylation status, the opposite response was observed with DHA treatment in the BT-474 luminal Her2-overexpressing cells, significantly enhancing Myc transcriptional activity and phosphorylation status. Although different responses in Myc activity existed between the two subtype-specific breast cancers, cell viability and apoptosis assays produced similar findings, revealing decreased cellular viability and increased early and late-apoptotic cellular status in response to DHA treatment. Additionally, dramatic decreases in wound healing and invasion index were observed in both cell lines with DHA supplementation compared to control treatments. Myc siRNA treatment rescued BT-474 cells from DHA treatment, confirming that DHA-induced alterations in apoptosis and invasion were initiated by Myc modification. Moreover, Myc knockdown in the MDA-MB-231 cell line displayed results similar to DHA treatment, consistent with our hypothesis that Myc activity is responsible for cellular survival and progression in subtype specific breast cancer. These results show that DHA is applying some of its anti-cancer effects by modifying Myc activity, inducing apoptosis and decreasing invasion in breast cancer, revealing new dietary targets that may enhance current therapeutics.

Citation Format: Michael Mouradian, Palvinder K. Bains, Amy M. Chattin, Ronald S. Pardini. Docosahexaenoic acid attenuates survival and progression in subtype-specific breast cancer by modifying Myc activity. [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 3165. doi:10.1158/1538-7445.AM2014-3165
Abstract 3170: Alternative NF-κB pathway activation enhanced by deficient TRAF3 in human papillomavirus (HPV)-associated head and neck cancer
Authors: Zhang Jialing , Cheng Hui , Yang Xinping , Maiorov Emine Guven , Keskin Ozlem , Gursoy Attila , Nussinov Ruth , Chen Zhong , Waes Carter Van
Published: May 2015

Head and neck squamous cell carcinoma (HNSCC) arises from the oral cavity, pharynx and larynx, and is the 6th most common cancer worldwide. The incidence of oropharyngeal SCC (OSCC) due to HPV is rising despite a decline in tobacco related HNSCC. However, the molecular mechanisms promoting HPV+ OSCC has not been clearly established. Recently, The Cancer Genome Atlas (TCGA) HNSCC project revealed a higher frequency of homozygous and heterozygous deletions in chromosome 14q32.32 associated with lower expression of the TNF receptor-associated factor 3 (TRAF3) gene, unique to HPV+ tumors. Deleterious nonsense and frame shift mutations of TRAF3 are also exclusively observed in HPV+ HNSCC. Structural interactions of TRAF3 with other signaling molecules were modeled three dimensionally using the PRISM algorithm (PRotein Interactions by Structural Matching). The interactions revealed that these TRAF3 mutations abolished key inhibitory interactions at the interface between receptors and NF-κB inducing kinase (NIK) in the alternative NF-κB pathway. Consistent with TCGA findings, we found varying TRAF3 expression in HPV+ HNSCC cell lines. Overexpression of TRAF3 in HPV+ cell lines suppressed the alternative NF-κB pathway and p52/p100 nuclear localization, and key target genes, while increasing expression of IFN genes implicated in anti-viral immunity. Further, overexpression of TRAF3 inhibited cell growth, colony formation, and migration, as well as sensitized cells to TNF-α and cisplatin induced cell death. Our data reveal that decreased TRAF3 promotes the malignant phenotype via alternative NF-κB signaling, and attenuates antiviral IFN expression, important in pathogenesis of HPV+ HNSCC.

Supported by NIDCD intramural project ZIA-DC-000016, 73, 74

Citation Format: Jialing Zhang, Hui Cheng, Xinping Yang, Emine Guven Maiorov, Ozlem Keskin, Attila Gursoy, Ruth Nussinov, Zhong Chen, Carter Van Waes. Alternative NF-κB pathway activation enhanced by deficient TRAF3 in human papillomavirus (HPV)-associated head and neck 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 3170. doi:10.1158/1538-7445.AM2014-3170
Abstract 3210: Supercritical extract of Azadirachta indica in chemoprevention of colon cancer
Authors: Khoogar Roxanne , Mukhophadyay Keya , Morris Jay , Soman Girish , Wargovich Michael
Published: May 2015

The leaves bark, and seed of Azadirachta Indica (Neem tree) have long been used in Ayurvedic medicine for health benefits. We have investigated a Supercritical CO2 Neem Extract (SCNE) in human colon cancer cell lines for chemopreventive potential.

A SCNE of leaf was procured from Nisarga Ltd, Sartara, India and subjected to HPLC and LC-MS to develop of signature of triterpenoids with potential cancer preventive activity. One compound, nimbolide, suggested to account for anti-proliferative activity against cancer cells was found to be present in the extract. The effects of the SCNE were determined on HT29 and HCT116 human colon cancer cell lines. The proliferative activity of HT29 and HCT116 cells were studied after 48 and 72 hours treatment with SCNE and Nimbolide at various concentrations. We also evaluated the effects of SCNE and nimbolide on cell cycle parameters, and examined relevant proteins associated with cancer cell proliferation. Cell cycle analysis was performed by flow cytometry and confirmed a pre-apoptotic peak in SCNE treated HCT116 cells. The modulation of apoptotic markers (intrinsic pathway: Bax, Bcl-2, and caspase-3, 9), proliferation pathway (cyclin D1) were studied at the protein and mRNA level. Treatment with SCNE and Nimbolide in a range from 8- 18 µg/ml resulted in dose -dependent growth inhibition in 48 and 72 hours. At these concentrations SCNE caused changes in cyclin D1. The induction of apoptosis by SCNE in HT29 and HCT116 was shown by caspase activation, cleavage of PARP, increased levels of pro-apoptotic protein Bax, and reduced levels of the anti-apoptotic protein Bcl-2. The effect of SCNE on colon cancer cell migration will be reported by scratch wound healing assay.

Overall, our data suggested that SCNE can suppress the growth of human colorectal cancer through induction of apoptosis. Current experiments are now focused on NF-kB and associated proteins as targets of SCNE inhibition.

Supported by grant P30CAO54174.

Citation Format: Roxanne Khoogar, Keya Mukhophadyay, Jay Morris, Girish Soman, Michael Wargovich. Supercritical extract of Azadirachta indica in chemoprevention of colon 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 3210. doi:10.1158/1538-7445.AM2014-3210
Abstract 3212: Ganoderma lucidum (Reishi) chemosensitizes EGFR-overexpressing inflammatory breast cancer cells to erlotinib
Authors: Suárez-Arroyo Ivette , Moraskie Michael , Cubano Luis A. , Martínez-Montemayor Michelle M.
Published: May 2015

Inflammatory Breast Cancer (IBC) is an aggressive cancer with a unique ability of forming tumor spheroids, which invade the dermal lymphatics causing the inflammatory phenotype. IBC cell lines and human tissues, overexpress Epidermal Growth Factor Receptor (EGFR), which has been associated with increasing IBC tumor growth rate, invasion and metastasis. Among the current therapeutic strategies for EGFR inhibition are the small molecule Tyrosine Kinase Inhibitors (TKIs). The high incidence of resistance to such therapies has greatly diminished their overall effectiveness, thus it seems feasible to identify agents that may be combined with these TKIs to provide a sustained response for IBC patients. For this reason, the purpose of this study is to investigate Reishi's therapeutic potential in IBC, focusing on the regulation of the EGFR signaling cascade and its contribution to the IBC cellular response when treated with the EGFR TKI, erlotinib. Our hypothesis is that Reishi sensitizes IBC cells to erlotinib therapy. EGFR-overexpressing SUM-149 IBC cells were treated with several concentrations of erlotinib, 0.025mg/mL Reishi, or both treatments simultaneously for 72h. Our results showed a synergistic effect when erlotinib and Reishi are used simultaneously after 72 hours of treatment. In order to study the molecular mechanism for the improved effects of the combination treatment we successfully developed an IBC erlotinib resistant cell line from the parental SUM-149 cells. Our data revealed that Reishi increases erlotinib sensitivity by inactivating AKT and extracellular signal-regulated kinase (ERK) signaling pathways. Taken together, our results provide evidence that Reishi chemosensitizes IBC cells to erlotinib therapy, highlighting its anti-IBC-therapeutic potential. This project was sponsored by NIH/NCI #1F31 CA174307 to ISA, Title V PPOHA US Department of Education #P031M105050 to UCC, NIH/RCMI #G12 MD007583 to UCC, NIH/INBRE #5P20 GM103475 to UPR/UCC and a research donation from the Commonwealth of Puerto Rico to UCC's University Center of Integral and Complementary Medicine (CUMIC)/MMM.

Citation Format: Ivette Suárez-Arroyo, Michael Moraskie, Luis A. Cubano, Michelle M. Martínez-Montemayor. Ganoderma lucidum (Reishi) chemosensitizes EGFR-overexpressing inflammatory breast cancer cells to erlotinib. [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 3212. doi:10.1158/1538-7445.AM2014-3212
Abstract 3219: Synergistic effect between Ganoderma lucidum (Reishi) and lapatinib in HER2+ inflammatory breast cancer cells
Authors: Feliz-Mosquea Yismeilin , Suárez-Arroyo Ivette , Cubano Luis A. , Martínez-Montemayor Michelle M.
Published: May 2015

Inflammatory Breast Cancer (IBC) is the most lethal and rare form of breast cancer. It is characterized by rapid progression, local and distant metastases, younger age of onset, and lower overall survival compared with other breast cancers. Although IBC, like non-IBC breast cancers, is a heterogeneous disease and can occur as any of the five molecular breast cancer subtypes, they are most commonly either triple negative; or ER-, PR- and HER2+ overexpressed. There is no specific IBC treatment; thus, it seems feasible to find a therapeutic for this deadly disease. Our published data demonstrates that Ganoderma lucidum (Reishi) inhibits the viability of the triple negative IBC SUM149 cell line, but not of MCF10A noncancerous mammary epithelial cells. Consequently, in this study, we aimed to investigate Reishi effects in the viability of the ER-, PR-, HER2+ IBC cells lines SUM190 and KPL4. Also we studied the effects of Reishi on IBC cells when treated in complement with the HER2 Tyrosine Kinase Inhibitor, lapatinib (Tykerb®). Our hypothesis is that Reishi chemosensitizes IBC cells to lapatinib therapy. Herein, SUM190 and KPL-4 cells were treated with increasing concentrations of lapatinib and/or Reishi for 24 or 72h. Our results demonstrate that SUM190 and KPL-4 cells are sensitive to Reishi treatment. The combination of lapatinib plus Reishi reduced cell viability of SUM190 cells to 70% when treated with a concentration of 0.5mg/mL Reishi for 24h. Also, our data shows a reduction in cell viability of 94% in KPL-4 cells treated with lapatinib plus Reishi for 72h. We are currently directing our efforts to continue studying the contribution of Reishi in IBC cells when treated with lapatinib after 24 or 72h. Our results provide evidence that Reishi inhibits cancer cell viability and chemosensitizes IBC cells to lapatinib therapy, highlighting its anti-IBC-therapeutic potential. We thank Dr. Kurebayashi (Kawasaki Medical School, Japan) for providing KPL-4 cells. This project was sponsored by NIH/NCI #1F31CA174307 to ISA, Title V PPOHA US Department of Education #P031M105050 to UCC, NIH/RCMI #G12MD007583 to UCC, NIH/INBRE #5P20GM103475 to UPR/UCC and a research donation from the Commonwealth of Puerto Rico to UCC's University Center of Integral and Complementary Medicine (CUMIC)/MMM.

Citation Format: Yismeilin Feliz-Mosquea, Ivette Suárez-Arroyo, Luis A. Cubano, Michelle M. Martínez-Montemayor. Synergistic effect between Ganoderma lucidum (Reishi) and lapatinib in HER2+ inflammatory breast cancer 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 3219. doi:10.1158/1538-7445.AM2014-3219