bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5215: CXCR6-mediated cellular and molecular events involved in breast cancer cell migration and invasion
Authors: Triplet Ashley , Sharma Praveen K. , Singh Rajesh , Lillard James W. , Grizle William E. , Singh Shailesh
Published: February 2011

Optimizing standard treatment modalities for breast cancer has improved the outlook for women afflicted with it. However, 40% breast cancer patients still die from the disease, primarily due to metastasis, which highlights the need for new therapies and better understanding of the mechanisms involved in disease progression. The reason(s) why breast cancer cells preferentially metastasize to lymph nodes, bone, brain, and lung is not fully understood. In this regard, chemokines and their corresponding receptor interactions in metastasis is an area of intense pre-clinical and clinical research. Analysis of breast cancer tissue micro-array in our lab demonstrated expression of CXCR6 by the breast cancer tissue. In confirmation with the TMA, breast cancer cell lines (MCF-7 and MDA-MB-231) also showed higher expression of CXCR6 in comparison to the normal transformed memory epithelial cell line (MCF-10A). We further investigated the role of CXCR6 in breast cancer cell migration and invasion, which are the key steps used by the cancer cells to achieve metastatic goals. Interestingly, breast cancer cells showed higher migratory and invasive potential towards the chemotactic gradients of CXCL16, which is the only natural ligand of CXCR6. This invasive and migratory potential was significantly inhibited after CXCR6 blockade. Our antibody array data show phosphorylation Src, FAK and ERK1/2 in breast cancer cells following CXCL16 treatment as compared to normal breast cancer cell. To determine the molecular mechanisms by which the CXCR6-CXCL16 axis mediates migration and invasion, inhibitors against Src kinase, FAK and ERK1/2 were used during the migration and invasion assay, which resulted in decreased migration and invasion. This suggests that Src, FAK, and ERK1/2 are required for CXCR6-mediated migration and invasion. Furthermore, an increase in F-actin polymerization was observed after CXCL16 treatment, which was significantly inhibited by the pharmacological inhibitors against Src, FAK, and ERK1/2, suggesting their potential role in CXCR6-mediated cytoskeletal rearrangement during breast cancer cell migration and invasion. Taken together, these results indicate clinical and biological significance of CXCR6-CXCL16 axis in breast cancer and targeting this axis may provide new therapeutic option.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5215.
Abstract 5216: An Akt-EphA2 reciprocal regulatory loop controls tumor cell migration and invasion
Authors: Miao Hui , Guo Hong , Petty Aaron , Sloan Andrew , Danielpour David , Basilion James , Cutter Jennifer , Sedor John , Cohen Mark , Mukherjee Amitava , Wang Bingcheng
Published: February 2011

EphA2 is a member of Eph family receptor tyrosine kinases that has been implicated in many types of human cancers. Perplexingly, both pro- and anti-oncogenic properties have been attributed to EphA2 kinase. We report here that a possible cause for this apparent paradox is diametrically opposite roles of EphA2 in regulating cell migration and invasion. While activation of EphA2 with its ligand ephrin-A1 inhibited chemotactic migration of glioma and prostate cancer cells, EphA2 overexpression promoted migration in a ligand-independent manner. Surprisingly, the latter effects required phosphorylation of EphA2 on serine 897 by Akt, and S897A mutation abolished ligand-independent promotion of cell motility. Ephrin-A1 stimulation of EphA2 negated Akt activation by growth factors and caused EphA2 dephosphorylation on S897. In human astrocytoma, S897 phosphorylation was correlated with tumor grades and Akt activation, suggesting that the Akt-EphA2 crosstalk may contribute to brain tumor progression.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5216.
Abstract 5224: Tracking the longitudinal physiological and vascular morphology changes in response to anti-angiogenesis treatment in breast tumors
Authors: Cao Ning , Cao Minsong , Liu Bo , Miller Kathy D. , Stantz Keith
Published: February 2011

VEGF and its receptor (VEGFR2 or flk1/KDR) play critical roles in tumor angiogenesis, which is essential for tumor growth and metastasis. The objective of this study is to track and quantify the longitudinal intra-tumor heterogeneous response to anti-VEGFR2 treatment using a combination of imaging modalities: dynamic contrast-enhanced CT (DCE-CT) and photoacoustic computed tomography (PCT). DCE-CT is used to measure vascular physiology and PCT is used to monitor its morphological changes based on its hemoglobin status within the tumor.

A cohort of athymic mice bearing MCF-7VEGF (MCF-7 cells transfected with VEGF gene) tumors were divided into three groups: a control group (received non-specific, rat immunoglobulin IgG1, i.v.), a low dose therapeutic group (30 mg/kg DC101, i.v.), and a high dose therapeutic group (150 mg/kg DC101, i.v.). Twenty-four hours prior to treatment, baseline DCE-CT and PCT scans were acquired. Successive scans were taken 2 days, 1 week, and 2 weeks post treatment to track both physiological and morphological changes. For the former, a two-compartmental kinetic model is fit to contrast enhanced curves on a voxel-by-voxel basis to determine tumor physiological parameters of perfusion (F), permeability-surface area (PS), fractional plasma volume (fp) and fractional interstitial space (fis). Maximum intensity profiles (MIPs) and hemoglobin profiles were extracted from PCT images.

The preliminary results show a dose dependent response to DC101 treatments. Two days post treatment, low dose reduced F by 36% and fp by 22%; high dose reduced F by 72% and fp by 39%. Tumor perfusion remained reduced two weeks post treatment, while fp in treated mice rebounded to similar levels as the control group after one week and exceeded the control group by week two. This decoupling of fp and F over time indicates a step-wise progressive nature to the formation of a vascular network and its functionality associated with the VEGF/VEGFR2 pathway. Photoacoustic results confirm the link between the changes in vascular morphology and physiology, and provide an in vivo link between vascular structure and function in response to anti-angiogenic therapy. Future work will focus on measuring the oxygen saturation levels and hemoglobin concentration in tumors by analyzing the spectroscopic information afforded by PCT.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5224.
Abstract 5227: Dynamic bioluminescent imaging and fluorescent imaging evaluation of vascular targeting agents combretastatin-A4P on brain tumor xenografts
Authors: Liu Li , Mason Ralph P. , Gimi Barjor
Published: February 2011

Bioluminescent imaging (BLI) is an inexpensive, high-throughput approach to assess tumor growth in small animals. Recent experiments show that vascular targeting agents, which disrupt the blood supply rapidly, cause reduction and delay in emitted light as assessed by the kinetics of BLI signal intensity in breast tumor.

We transfected 9L rat glioma cells to express luciferin under the HRE promoter, and transduced these cells with a retrovirus to constitutively express GFP and mCherry. Different numbers of 9L-3HRE-luc/GFP/mCherry cells were implanted in the lower back regions of nude mice. When tumors reached about 3 to 7 mm diameter, they were imaged first with Fluorescence imaging (FLI) to detect GFP and mcherry signal. Then, 80 µl luciferin (40 mg/kg) was administered S.C and a BLI time course was acquired over 50 mins using a Caliper Xenogen Spectrum. Combretastatin-A4P (CA4P) was then administered IP (150 mg/kg in 100 μl saline) and FLI and BLI repeated following the administration of fresh luciferin 2 h and 4h later. The processes were repeated at the 24 h and 96 h time points.

We examined the effect of CA4P on 9L-3HRE-luc/GFP/mCherry that are implanted as S.C xenografts in nude mice, and found highly reproducible curves under control conditions. 2h following CA4P, the BLI signal was reduced by 90%, 4h later, some recovery was observed. After 24 h, the light emission continued to recover. After 96 h, signal was observed mainly from the periphery of larger tumors, probably owing to central necrosis. Notably, the GFP and mCherry fluorescent signals were slightly reduced after 2 h, further declined after 4 h, and GFP signal was completely eliminated after 24 h, attributable to the vascular hemorrhage absorbing GFP. Similarly, after 96 h, both GFP and mCherry signal could be detected in the periphery of larger tumors. Three tumors were developed under similar conditions and examined simultaneously, which provides a valid assessment of vascular shutdown. This technique can be utilized to optimize future therapeutic drug dosage and combinations.

Support: NIH R01 EB007456 and NCI SAIRP U24CA126608

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5227.
Abstract 5229: In vivo bioluminescence imaging of intracranially implanted glioma cells and opening of the blood-brain-barrier in mice
Authors: Bode Julia , Huelper Petra , Lakomek Max , Kugler Wilfried
Published: February 2011

Malignant gliomas (glioblastoma multiforme and anaplastic astrocytoma) occur more frequently than other types of primary central nervous system tumors. Their combined incidence is 5-8/100.000 population.

Glioblastoma multiforme is characterized by an intrinsic resistance to radio- and chemotherapy, which is mainly responsible for the poor prognosis of patients with high grade glioma. The median reported survival is less than 1 year.

Pharmaceutical treatment of brain malignancies is restricted due to the poor penetration of drugs across the blood-brain-barrier (BBB). The intracarotid bolus administration of short chain alkylglycerols has been reported to be an effective and low toxic strategy to increase the transfer of various drugs to both the normal brain and brain tumors in mice. Thus we administrate short chain alkylglycerols via an intracarotid bolus injection and image the additional administration of a fluorescence compound.

In order to investigate the effect of the tumor treatments we had to establish a new imaging model for in vivo experiments.

The glioma cells U87MG and U373MG were lentivirally transfected with luciferase and implanted stereotactically into the brain of eight weeks old immundificient nude mice.

We injected the substrate firefly luciferine intraperitoneally and imaged the mice every two days from day 14 after injection of the cells into the brain. We took out the brains, cut them into slices and stained the slices with H&E. We calculated the detected bioluminescence signals of the tumors and we saw a correlation between the signals and the size of the tumors in the H&E stainings.

For the BBB opening we injected 90mM 2-O-hexyldiglycerol into the internal carotid artery. We administered fluorescence labeled compounds and imaged the brain tissue to detect the injected compounds.

We succeeded to open the BBB via administration of alkylglycerols. In addition we established a model for the detection of brain tumor growth. We imaged and measured the growth of brain tumors with the help of bioluminescence imaging in mice in vivo and now we can use this tool to measure the outcome of our treatments with anti cancer drugs.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5229.
Abstract 5235: Fluorometric imaging system for visualizing submillimeter level tumors using an ultrathin endoscope
Authors: Morimoto Yuji , Muraoka Miho , Miyazaki Kozo , Kumagai Michiaki , Horie Souta , Fukushima Shigeto , Matsumura Kouji , Matsuo Hirotaka , Shinomiya Nariyoshi , Nishiyama Nobuhiro , Kataoka Kazunori
Published: February 2011

[Background and purpose]

Direct observation of pathological lesions by a relatively noninvasive approach is important for understanding pathophysiological mechanism and for assessing the objective response to therapeutic interventions. On the other hand, it is very difficult to perform direct and relatively noninvasive observation of minimal lesions inside a diseased model using a small live animal. However, recent technological developments in imaging, especially significant progress in fluorescent methods and in endoscopic examinations, have enabled detection and tracing of micro-lesions inside small animal models.

We have developed a fluorescence imaging technique using high-resolution ultrathin endoscope, which is accessible to hollow organs of small animals. In addition, we have established an orthotopic rat bladder cancer model in which the cancer cells express a fluorescent protein. The aim of this study was to establish a high-resolution imaging method for visualizing the vascular distribution and mucosal morphology of a bladder tumor.

[Materials and Methods]

GFP-expressing bladder tumor in a rat orthotopic model:

Firstly, we established a cell line with stable integration of GFP constructs that were produced by transfection of pTurboGFP into rat bladder cells (AY27). The stable AY27+tGFP populations were purified by repeated selections using Geneticin (G418). AY27-tGFP cells were implanted in the bladders of female Fischer F344 rats, resulting in development of submillimeter-sized bladder tumors.

Development of fluorescence ultrathin endoscopy:

We developed a fluorescence ultrathin endoscopic system installed with a 180 W Xenon lamp with excitation filters and 3CCD camera with emission filters. The outer diameter of the ultrathin endoscopic fiber is only 0.8 mm, but its number of pixels reaches 15,000, resulting in a spatial resolution of 50μm.

[Results]

Fluorescence from AY27-tGFP cells implanted in the bladder was clearly visualized by the ultrathin endoscope placed in the bladder by the transurethral approach. Furthermore, when fluorescence-probed tumor-directive micelles were injected (i.v.) into the animals as a model of drug delivery, fluorescence from the probe agents was visualized in accordance with tumor distribution. In both cases, we detected fluorescence from tumors of less than 1 mm in size.

[Conclusion]

We established an orthotopic GFP-tumor rat model using a GFP-expressing tumor cell line (AY27-tGFP). Submillimeter tumors in this model were visualized by the newly developed ultrathin endoscopic system.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5235.
Abstract 5246: Use of near infrared fluorescence imaging to determine tumor burden in genetically engineered mice
Authors: Yoshikawa Kazuhiro , De Velasco Marco A. , Tanaka Motoyoshi , Miyazaki Yuka , Ushida Hiroshi , Asahi Chiori , Nishio Kazuto , Uemura Hirotsugu
Published: February 2011

Tumor imaging in mice revolutionized the ability to detect and quantify tumor burden in pre-clinical studies. However, this technology has been limited for the most part to the use of cancer cell lines manipulated to express specific fluorescence or bioluminescence in xenograft models. Recent advances in the generation of genetically engineered mouse (GEM) models have resulted in notable improvements as platforms for pre-clinical models. Variables such as initial tumor detection, size and location have influenced and limited objectivity in study design and interpretation. In this study we introduce a new modality for determining tumor burden using a commercially available recombinant EGF polypeptide labeled with a near infrared (NIR) fluoroprobe to identify and quantify tumor burden in a knockout mouse model. We evaluated the sensitivity and accuracy EGF-labeled IRDye using our prostate specific PTEN conditional gene-targeting mouse model using intact and castrated mice.

Mice were administered the EGF-labeled IRDye by tail vein injection 72 h prior to imaging in vivo imaging. The genitourinary tact (GUT), consisting of the prostate, urethra, bladder, seminal vesicles and coagulating gland, was collected, weighed, fixed in formalin and imaged and cryosectioned. EGFR expression was detected by immunohistochemistry and expression levels were measured by image analysis. To determine tumor burden, adjacent cryosections were analyzed by NIR fluorescence and by histology for size and distribution of tumor glands in H&E stained slides.

Closed abdomen in vivo imaging was not possible due to low signal and interference of the preputial gland. However, prostates surgically exposed revealed the presence of tumor nests in the prostate as small as 0.2 mm. Furthermore, formalin fixed GUTs retained fluorescence and were imageable up to 48 h post-fixation. EGFR overexpression was found to be concentrated in cancerous glands and expression was directly proportional to the NIR fluorescent signal. Tumor burden determined by GUT weight correlated poorly to NIR expression in castrated mice. Our data shows that EGF-labeled IRDye NIR imaging is a precise and sensitive method for the determination of tumor burden; however, at the present time, this technology is limited to invasive imaging or ex vivo applications. This study is the first to use NIR fluorescence to measure prostate tumor growth and response in GEM and thus provides a feasible alternative to conventional methods.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5246.
Abstract 5254: NLRR family proteins play distinct roles in deciding cell fate and affect the clinical outcome in neuroblastomas
Authors: Takatori Atsushi , Hossain MD. Shamim , Akter Jesmin , Nakagawa Atsuko , Suenaga Yusuke , Nakamura Yohko , Ohira Miki , Ozaki Toshinori , Nakagawara Akira
Published: February 2011

We have previously generated neuroblastoma (NB)-prone oligo-capping cDNA libraries from primary neuroblastomas and identified novel important genes. Among those, we selected human neuronal leucine-rich repeat protein (NLRR) family genes for understanding of their functional roles in biology and clinical behavior of NB. In our previous observations, the expression level of NLRR1 mRNA is significantly higher in unfavorable NBs than favorable ones and its high expression is associated with poor clinical outcome, whereas that of NLRR3 is significantly high in favorable NBs in association with good prognosis. These findings suggested that NLRR1 and NLRR3 may have a different and opposite role in regulating cell growth and differentiation in NB. On the other hand, NLRR2 is similarly expressed in both subsets of NB. However, their real function in NB still remains elusive. Our present studies showed that cell proliferation was increased by overexpression of NLRR1 with enhanced EGF and IGF signals. NLRR1 proteins localized in cholesterol-rich lipid raft microdomain of cellular membrane and its expression altered the membrane distribution of EGFR and IGF-1R. Treatment of methyl-β-cyclodextrin to deplete membrane cholesterol and disrupt lipid rafts diminished the effect of NLRR1 on EGFR, suggesting that NLRR1 accelerates growth signaling pathway through lipid raft microdomains. Transcriptional study revealed that stimulation of EGF and IGF signaling enhanced the transcriptional activity of MYCN gene and MYCN in turn transactivates NLRR1 transcription, suggesting that NLRR1 and MYCN, both of which are poor prognostic factors in NBs, form a positive feedback loop via EGFR and IGF-1R. On the contrary, we observed opposite results in the study on NLRR3. The transcription was negatively regulated by MYCN with Max and Miz-1. Interestingly, enforced expression of NLRR3 induced neuronal differentiation and siRNA-mediated knockdown of NLRR3 reduced retinoic acid-induced neuronal differentiation in NB cells. Quantitative PCR also showed the inverse relationship between NLRR3 and MYCN in primary NB tissues. The combination of low NLRR3 and low Miz-1 expression or that of low NLRR3 and high MYCN expression was significantly associated with a poor prognosis. The current study on NLRR2 so far indicated its involvement in endoplasmic reticulum-stress response. Collectively, our results suggest that the transcriptional regulation varies between NLRR family genes and they play different roles in cell fate decision and contribute to the clinical outcome of NBs.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5254.
Abstract 5259: Application of HT-RNAi-based functional genomics in neuroblastoma
Authors: Gonzales Irma M. , Arora Shilpi , Khan Javed , Azorsa David O.
Published: February 2011

Neuroblastoma is the most common extra-cranial solid cancer in children. About 70% of patients with neuroblastoma have metastatic disease at the time of diagnosis. Children with favorable disease characteristics and younger than one year of age (stages I and II) have a higher likelihood of disease-free survival. However, older children with advanced disease (stages III and IV) have only a 30% chance of survival even with aggressive therapy. Additionally, amplification of the MYC-N gene has been associated with stage III and IV tumors, thus establishing it as a poor prognostic marker. Current treatment primarily consists of combinations of surgery, radiation, and chemotherapy. Newer regimens also include the topoisomerase I inhibitor topotecan, which has been found to be effective against recurrent disease. In order to identify “Achilles heel” targets that modulate growth of neuroblastoma, we applied a functional genomics approach using High Throughput RNA interference (HT-RNAi). Furthermore, by treatment of cells with low dose topotecan, we hope to identify genes the can improve the response to this current therapy. We first identified optimal siRNA transfection conditions for the four neuroblastoma cell lines; MYC-N amplified, IMR-5 and IMR-32 and MYC-N non-amplified, SK-N-AS and NB-EB. Since these cell lines can be difficult to transfect with siRNA, several cationic lipids were tested at varying lipid:siRNA ratios to obtain the optimal reagent and ratio for each cell line. Next, we determined the optimal low dose (IC10-30) of topotecan for each cell line. Following these optimizations, HT-RNAi assays were conducted on the four neuroblastoma cell lines using a kinase siRNA library targeting 572 kinases and a siRNA library targeting 532 apoptosis associated genes treated with vehicle or low dose topotecan. These HT-RNAi screens identified genes whose silencing decreased neuroblastoma cell growth and genes whose silencing improved topotecan response. We are currently in the process of validating these results. After validation, we hope to develop these molecular targets into innovative therapeutics for children affected with this disease.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5259.
Abstract 5260: Bortezomib and HDAC inhibitor PCI-24781 combination therapy for neuroblastoma
Authors: Currier Erika A. , Illenye Sharon A. , Ashikaga Takamaru , Lescault Pamela J. , Bond Jeffrey P. , Sholler Giselle L. Saulnier
Published: February 2011

Background: Neuroblastoma is an aggressive solid tumor with poor prognosis. Novel therapies are needed to help increase the survival and quality of life for affected children. It has been previously shown that bortezomib results in apoptosis in neuroblastoma both in vitro and in vivo. PCI-24781 is a novel pan-HDAC inhibitor which has been shown to be synergistic with bortezomib in lymphoma models. We hypothesize the combination of bortezomib with PCI-24781 would increase cytotoxicity to neuroblastoma.

Methods: Several established neuroblastoma cell lines as well as patient-derived primary neuroblastoma cultures were grown in 96-well plates and treated with bortezomib and PCI-24781, both separately and in combination for 48 hours. Cell viability was assessed by calcein AM assays and IC50's and drug synergy was determined at each cell line tested. mRNA was collected from drug treated cell lines and effects on cell signaling was evaluated using U133 mRNA expression arrays and Ingenuity analysis. Cell lysates prepared from drug treated cells were evaluated by western blot for caspase-3 and PARP cleavage. Nude mice were injected with 107 SMS-KCNR cells subcutaneously in the left flank. Mice were treated with daily doses of either 0.5 mg/kg bortezomib, 12.5 mg/kg PCI-24781, or the combination. Mice tumors were imaged using the IVIS lumina imager twice per week and caliper measurements were also obtained weekly until tumor max was reached.

Results: All neuroblastoma cell lines and patient cells tested showed sensitivity to bortezomib and PCI-24781 treatment in calcien AM cell viability assays with IC50's for bortezomib less than 50nM and IC50's for PCI-24781 less than 500nM. The combination of bortezomib and PCI-24781 was more cytotoxic than either drug alone. Cells treated with combined treatment showed an increase in caspase-3 and PARP activity confirming apoptosis. Expression analysis showed differential expression in the Notch and Wnt pathways between drug treated and vehicle treated cells. The xenograft models showed a significant decrease in tumor volume in mice treated with both bortezomib and PCI-24781 when compared to the single agent treatment groups or to the control group. Conclusions: Bortezomib and PCI-24781 inhibit neuroblastoma growth both in vitro and in vivo to a greater extent than either drug alone. The mechanism of action is currently being investigated further but certainly involves caspase-3 mediated apoptosis. This combination therapy appears to be effective and well tolerated in the mouse model and would be a novel therapy for neuroblastoma.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5260.
Abstract 5263: An RNAi screen of the protein kinome identifies CHEK1 as a therapeutic target in neuroblastoma
Authors: Cole Kristina A. , Huggins Jonathan , Laquaglia Michael , Diskin Sharon , Attiyeh Edward F. , Hulderman Chase , Han Miryung , Bosse Kristopher , Jagannathan Jayanti , Mosse Yael P. , Mayes Patrick , Maris John M.
Published: February 2011

Background High-risk neuroblastoma is a pediatric cancer that is often fatal despite intense multimodality therapy associated with significant late effects for those who survive. Therefore, patients will benefit from treatment strategies that rationally exploit signaling pathways for which a tumor cell is selectively dependent. In an effort to identify novel therapeutic targets, we performed a comprehensive loss of function screen of the protein kinome in neuroblastoma cell models.

Methods Using a validated siRNA library which targets the human protein kinome, we transiently transfected individual kinase siRNAs into four neuroblastoma cell lines and measured substrate adherent growth. We then ranked the top 100 most potent kinase siRNAs per cell line and integrated the lists to identify kinases with inhibition in multiple lines.

Results Thirty targets emerged as potent in at least three of the four neuroblastoma cell lines. They were enriched for kinases involved in the cell cycle and mitosis. This select group was tested with an independent set of more specific siRNA pools in an expanded panel of eight neuroblastoma cell lines. The strikingly most potent to emerge from the secondary screen was the cell cycle checkpoint kinase CHEK1. CHEK1 knockdown appeared cytotoxic, often by 24 hours, coinciding with complete CHEK1 protein knockdown and an emergence of a substantial apoptotic fraction. As further validation of the siRNA results, pharmacologic inhibition with SB21807, a relatively specific CHEK1 inhibitor, showed cytotoxicity in 7 of 8 neuroblastoma cell lines with a median IC50 of 430 nM (range 62-677 nM), while the non-neuroblastoma lines RPE-1, DOAY and MCF7 cells were resistant with a median IC50 of 2609 nM (1066 - 2898 nM). The mechanism of selective inhibition in neuroblastoma is unclear as we did not identify mutations in the coding exons of CHEK1. Western blotting revealed, however, that CHEK1 is constitutively phosphorylated at the Ser 296 residue in 8/8 neuroblastoma cell lines and 10/16 primary tumors samples, but not in controls.

Conclusion We have identified CHEK1 as a potential therapeutic target in neuroblastoma and are currently extending our work to understand the mechanism of this tumor's apparent selective sensitivity to CHEK1 inhibition. As CHEK1 inhibitors are currently in phase I/II clinical trials as chemosensitizers, we are also focused on determining the in vivo efficacy of combination CHEK1 inhibition with chemotherapy in neuroblastoma.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5263.
Abstract 5267: Mammary stromal fibroblast activity is modulated by HoxC6: impacts on growth and metastatic potential
Authors: Smith Ashley M. , Neumann Carola A. , Moreno Carlos S. , Maroulakou Ioanna G. , Rosenzweig Steven A. , Spyropoulos Demetri D.
Published: February 2011

Crosstalk between mammary epithelium and stromal cell types, including fibroblasts and adipocytes is 1) essential for mammary gland development 2) likely impacts breast cancer potential and 3) significantly impacts the growth, invasion and metastasis of tumor cells. Although well recognized, molecular mechanisms important in stromal-epithelial crosstalk are still poorly understood. The purpose of this study is to better elucidate one potentially significant mechanism involving stromal fibroblast to epithelial paracrine signaling in the mammary gland. We previously demonstrated that HoxC6 was required for mammary epithelial cell growth and ductal branching morphogenesis using mouse models and 3-dimensional in vitro culture of human mammary epithelial cell lines. We also demonstrated that constitutive ErbB2/Her2/Neu receptor activation in epithelium of transgenics is sufficient to rescue HoxC6 deficiency in epithelial growth but not branching. Significantly, this model also shows that HoxC6 null stroma facilitates tumor metastasis. Here, we focus attention on stromal fibroblasts (vimentin positive cells) from wild type and HoxC6 null prepubertal mouse thoracic mammary glands (TMGs). When cultured in vitro, HoxC6 null TMG fibroblasts display multiple filopodia and extended cellular processes that could indicate an activated myofibroblast phenotype. In contrast, cultured wild type TMG fibroblasts appear quite similar to mouse embryonic fibroblasts. Quantitative comparisons of gene expression patterns in isolated fibroblasts indicate that HoxC6 null fibroblasts are more activated than wild type fibroblasts. Activation markers studied include alpha smooth muscle actin (ACTA2/SMA), S100A4 (Fsp1) and Wnt1-induced secreted protein 1 (Wisp1). Since estrogens are know to repress HoxC6 expression and are implicated in modulating paracrine stromal-to-epithelial interactions, stromal fibroblasts were isolated and cultured under estrogen-free conditions and treated with 17beta-estradiol, bisphenol A and vitamin D. In addition, stromal fibroblasts were co-cultured with mammary epithelial stem cells and stem-like mammary tumor cells to study the impact of stromal fibroblast HoxC6 status on epithelial cell activities. The results obtained will be discussed as they pertain to stromal-to-epithelial signaling in mammary gland development and in the growth, invasion and metastasis of tumor cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5267.
Abstract 5272: Endothelin-1 enhances cell migration in human chondrosarcoma cells through PI3K signaling pathways
Authors: Wu Min Huan , Tang Chih-Hsin , Huang Chih-Yang , Lin Tzu En
Published: February 2011

Tumor malignancy is associated with several features such as proliferation ability and frequency of metastasis. Endothelin-1 (ET-1), the most potent vasoconstrictor, and plays a crucial role in the migration and metastasis of human cancer cells. We found that treatment of human chondrosarcoma cells (JJ012 cells) with ET-1 increased migration activity and the expression of matrix metalloproteinase (MMP)-13. Pretreatment of cells with phosphatidylinositol 3-kinase (PI3K), AKT and mammalian Target of Rapamycin (mTOR) inhibitor reduced ET-1- mediated cell migration and MMP-13 expression. In addition, NF-kappaB inhibitor (PDTC) or IkappaB protease inhibitor (TPCK) also inhibited ET-1 -mediated cell migration and MMP-13 up-regulation. Stimulation of cell with ET-1 induced PI3K, AKT and mTOR phosphorylation. Furthermore, the ET-1-mediated increasing kappaB-luciferase activity was inhibited by the specific inhibitor of PI3K, Akt and NF-kB cascades. Taken together, these results suggest that the ET-1 activate PI3K/AKT/mTOR, which in turn activates IKKalpha/beta and NF-kappaB, resulting in the activations of MMP-13 and contributing the migration of human chondrosarcoma cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5272.
Abstract 5276: CXCL7/CXCR2 axis and invasion
Authors: Yu Minghuan , Kosir Mary A.
Published: February 2011

Introduction: The CXCL7/CXCR2 axis interfaces with matrix molecules whereby the chemokine CXCL7 binds heparan sulfate and the G-protein-linked receptor CXCR2. CXCL7-transfected breast cells acquire the ability to invade through Matrigel. The purpose of this study was to test the effect of inhibition of heparan sulfate binding and CXCR2 binding on invasion.

Methods: Stably transfected breast cells (MCF10AT) were tested with CXCL7-siRNA, and a specific CXCR2 antagonist (SB225002). Inhibition was verified by real time PCR and heparanase ELISA assay. Matrigel invasion assays were performed.

Results: SB225002 and CXCL7 siRNA blocked invasion through Matrigel as compared to controls (p<0.01), and CXCL7 siRNA significantly decreased heparanase activity (p<0.01).

Conclusion: Interfering with either heparan sulfate binding by CXCL7 or its binding to CXCR2 leads to decreased invasion through Matrigel.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5276.
Abstract 5277: S100A7 modulates EGFR signaling in estrogen receptor negative breast cancer cell lines
Authors: Qamri Zahida , Nasser Mohd W. , Abdussamad Abdalla , Lill Nancy , Ganju Ramesh K.
Published: February 2011

Purpose of Study: S100A7 (psoriasin) is highly expressed in estrogen receptor alpha negative (ER-α−) and EGFR+ basal like phenotype, especially triple negative, that have been associated with worse prognosis and outcome. However, not much is known about the role of S100A7 in promoting tumorigenesis of ER-α− and EGFR+ breast cancers. We hypothesize that S100A7 secreted by tumor cells may modulate tumor growth by binding to receptor for advanced glycation end-products (RAGE) and activating EGFR in ER-α−. In the present study, we analyzed recombinant S100A7-mediated EGFR signaling that enhances breast tumorigenesis and metastasis in ER-α− cells.

Methods: We examined the effect of recombinant S100A7 on cell migration and motility of ER-α− MDA-MB231 and MDA-MB453 breast cancer cell lines. In addition, we analyzed S100A7-mediated signaling mechanisms by monitoring changes in phosphorylation and expression of various signaling molecules by Western blot, immunoprecipitation and immunofluorescence. We also co-transfected HEK-293 with EGFR and RAGE plasmids to analyze crosstalk between S100A7/RAGE and EGF/EGFR in these cells.

Results: We observed high expression of RAGE in ER-α− MB-231 and MB-453 breast cancer cell lines by FACS analysis. We have also shown that S100A7 is secreted in the conditioned media of ER-α− MB-231 overexpressing S100A7 cells. Recombinant soluble S100A7 was shown to induce cell migration and wound healing in MB-231 and MB-453 cells. These effects were mediated through RAGE, as RAGE neutralizing antibody blocked S100A7-mediated functional effects. To further analyze the molecular mechanism of S100A7-mediated signaling, we observed that recombinant S100A7 transactivates EGFR. Further analysis of molecular mechanism revealed that S100A7 enhances AKT and Erk phosphorylation as well as increases association between Jab-1 and EGFR. The crosstalk between S100A7/RAGE and EGF/EGFR was further confirmed by co-transfecting HEK-293 cells with EGFR and RAGE. Recombinant S100A7 was shown to induce EGFR phosphorylation in these transfected cells, confirming S100A7 may transactivate EGFR by binding to RAGE. Conclusion: These studies reveal that S100A7 may enhance metastatic potential of ER-α− cells by binding to RAGE and activating EGFR signaling. Furthermore, these studies may help in the development of S100A7/RAGE as novel therapies target for ER-α− breast cancer, especially triple-negative and EGFR+ basal-like cancers, which are highly aggressive and difficult to treat.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5277.
Abstract 5278: Inhibition of tumor cell migration and metastasis by the GPR4 receptor
Authors: Castellone Reid D. , Leffler Nancy R. , Yang Li V.
Published: February 2011

GPR4, a G protein-coupled receptor, has been shown to function as a proton sensor. Within tumor microenvironments, the interstitial acidic pH may activate GPR4 to regulate the behavior of tumor cells, blood vessels, and other cells. The expression of GPR4 is detected in some tumor cells, and it is the focus of this study to investigate the effects of acidosis/GPR4 signaling on tumor cell migration and metastasis. B16F10 melanoma cells and TRAMP-C1 prostate cancer cells, genetically engineered to overexpress GPR4 or the control vector, were subject to a series of cell motility assays. Transwell and wound closure assays were performed to examine cell migration, and it was observed that upon GPR4 activation in an acidic pH, cell migration was inhibited two to four fold in comparison to the vector control. These same trends were observed for invasion assays, in which Matrigel or a HUVEC endothelial monolayer was applied to the top of each transwell filter. To elucidate the molecular mechanism by which acidosis/GPR4 mediated motility inhibition occurs, G-LISA RhoA activation assays were used and it was observed that upon GPR4 activation in an acidic environment, the levels of activated RhoA, when compared to the vector control, were increased two and three fold. Furthermore, activation of Rho by its activator CN01 in B16F10 and TRAMP-C1 tumor cells recapitulated the acidosis/GPR4 induced motility inhibition phenotype, while inhibition of Rho reversed such phenotype. To examine the biological effects in vivo, B16F10 cells were injected into C57BL/6 female mice through tail veins. Pulmonary melanoma metastasis was inhibited by greater than 80% in GPR4 overexpressing B16F10 cells in comparison to the vector control. Upon treatment with the Rho activator CN01 in culture, the phenotype of the B16F10 vector cells paralleled that of the GPR4 overexpressing cells as metastasis was inhibited by more than 90%. These findings suggest that GPR4 activation by acidic pH plays a role in the inhibition of migration and invasion in B16F10 and TRAMP-C1 tumor cells, and the activation of Rho is at least partly responsible for this phenotype on the molecular level.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5278.
Abstract 5279: LPS-TLR4 binding increases metastatic potential of colorectal cancer cells
Authors: Hsu Rich YC , Chan Carlos HF , Spicer Jonathan D. , Rousseau Mathieu C. , Perera Rushika , Giannias Betty , Rousseau Simon , Ferri Lorenzo E.
Published: February 2011

Objective: Infectious complications in colorectal cancer patients undergoing curative resection are associated with cancer recurrence, but the exact mechanism is not well understood. Toll-like receptor 4 (TLR4) is the sole receptor for lipopolysaccharide (LPS), a gram-negative bacterial antigen involved in such infectious complication. TLR4 expression has been found on several cancer types. We hypothesize that LPS promotes cancer cell metastatic potential via TLR4 signaling in colorectal cancer cells.

Methods: We compared the effects of LPS stimulation on two well-characterized colorectal cancer cell lines: Caco-2 (non-metastatic) and HT-29 (metastatic). Flow cytometry was performed to detect cell surface TLR4 and various integrin expression. Cancer cells were incubated with or without 0.1 µg/mL LPS for 4 hours. In vitro static adhesion assay was used to study the ability of cell adhesion to extracellular matrix proteins (fibronectin, collagen I and collagen IV). Intravital microscopy, a technique used to study microcirculation in vivo, was carried out to observe dynamic attachment of intra-splenic injected cancer cells to C57/BL6 mouse liver sinusoids.

Results: Caco-2 cells lack TLR4 receptors and are unresponsive to LPS treatment. In contrast, HT-29 cells were shown to express TLR4 receptors and HT-29 responds to LPS by a 2-fold increase of β1, but not α5, integrin expression in LPS-treated HT-29 cells (Mean Fluorescence Intensity:1041 vs. 458(untreated)). LPS-treated HT-29 cells showed enhanced attachment to fibronectin (1.6-fold; p < 0.01), collagen I (3-fold; p < 0.01) and collagen IV (2-fold; P < 0.01) relative to controls in vitro. Similarly, adherence of HT-29 cells to hepatic sinusoids in vivo was increased by LPS treatment (2 fold; p < 0.05).

Conclusion: These data suggest that LPS-TLR4 signaling in colorectal cancer cells increases the metastatic potential. LPS-TLR4 mediated signaling cascade may be a valuable therapeutic target for the prevention of cancer metastasis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5279.
Abstract 5282: TRPM7, a novel regulator of human nasopharyngeal carcinoma cells Ca2+ influx and migration
Authors: Chen Jian-Peng , Luo Rong-Cheng , Li Rong , You Chang-Xuan
Published: February 2011

Ion channels are involved in various physiologic and pathologic processes, including the migration of tumor cells that is required for metastasis. To determine whether transient receptor potential melastatin 7 (TRPM7) Ca2+ channels play an important role in the migration of tumor cells, we examined the potential function of TRPM7 channels in the migration of 5-8F human nasopharyngeal carcinoma cells. The migratory potential of the cells was impaired by TRPM7 inhibitors (La3+, 2-APB), and their migration was reduced significantly after either TRPM7 was silenced or extracellular Ca2+ was removed. Conversely, addition of TRPM7 activator (BK) promoted the migration of 5-8F cells. Furthermore, the Ca2+ influx regulated by TRPM7 activated release of Ca2+ stores by a calcium-induced calcium release mechanism. This study suggests, first, that Ca2+ influx is required for the migration of human nasopharyngeal carcinoma 5-8F cells. Second, and more importantly, it identifies TRPM7 as a novel potential-regulator of the Ca2+ influx that allows migration of 5-8F cells. TRPM7, therefore, might have potential as a prognostic indicator and as a therapeutic target in cancer.

Keywords: TRPM7; nasopharyngeal carcinoma cells; 5-8F; Ca2+ influx; Ca2+; migration.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5282.