bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 519: Differential expression of basement membrane type IV collagen α chains as a prognostic factor of extrahepatic bile duct carcinoma
Authors: Karashima Ryuichi , Hirashima Kotaro , Iyama Ken-ichi , Baba Yoshifumi , Sado Yoshikazu , Ninomiya Yoshifumi , Takamori Hiroshi , Baba Hideo
Published: February 2011

Objective: The destruction of the basement membrane is the first step of cancer cell invasion and metastasis. Type IV collagen is a major component of the basement membrane, and is composed of six genetically distinct α(IV) chains: α1(IV) to α6 (IV). The loss of α5/α6(IV) chains from the epithelial basement membrane at the early stage of cancer cell invasion has been reported in several types of cancers. However, the expression of α5/α6(IV) chains in extrahepatic bile duct carcinoma remains unclear. The expression status of α5/α6(IV) chains in extrahepatic bile duct carcinoma was examined.

Methods: The expression of α(IV) chains, p53, and Ki-67 were examined by using immunohistochemical method in 36 surgical specimens of extrahepatic bile duct carcinoma. The relationship between clinicopathological parameters of 36 patients and the results of immunohistochemical staining was statistically examined using χ2 test or Fisher's exact test. The overall cancer-specific survival was estimated using the Kaplan-Meier method, and the log-rank test was used to evaluate the statistical significance of the differences.

Results: In the normal bile duct mucosa of all examined specimens, both α1/α2(IV) and α5/α6(IV) chains were stained in the basement membrane of the surface columnar epithelium in a continuous linear pattern, while both α3(IV) and α4(IV) chains were not stained. The α1/α2(IV) and α5/α6(IV) chains were also expressed in the basement membrane of smooth muscle cells of the muscularis propria and large blood vessels. In extrahepatic bile duct carcinoma, α5/α6(IV) chains disappeared partially or completely around the tubular cancer cells in 25 of 36 specimens. All of tumors expressed α5/α6(IV) chains were also positive for α1/α2(IV) chains. The expression of α5/α6(IV) chains were significantly correlated to T classification and TNM staging, and inversely correlated to p53 expression. No relationship was observed between the expression of α5/α6(IV) chains and Ki-67 index. The patients with loss of α1/2(IV) and α5/6(IV) chains demonstrated significantly poorer prognosis than those with positive α1/2(IV) chains, irrespective of α5/6(IV) expression.

Conclusion: The loss of α1/2(IV) and α5/α6(IV) chains might be a useful prognostic factor for extrahepatic bile duct carcinoma.

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 519.
Abstract 5192: GEP100 binding to HER2 receptor promotes lung cancer invasion and metastasis
Authors: Toshi Menju , Hashimoto Shigeru , Hashimoto Ari , Date Hiroshi , Sabe Hisataka
Published: February 2011

HER2 overexpression is well known to be a poor prognostic factor in some of epithelial tumors, including breast cancer and lung cancer. Previous reports have shown that GEP100-Arf6-AMAP1 pathway is pivotal for significant population of breast cancer invasion originated from EGFR signaling. Here we have examined the involvement of this pathway in HER2 signal-induced lung cancer invasion and / or metastasis.

Co-immunoprecipitation for the reconstitutive expression of both HER2 and GEP100 or ARNO, the other ArfGEF for Arf6, showed that GEP100, but not ARNO, bound HER2 receptor through its pleckstrin homology (PH) domain. The stimulation of reconstitutively expressed cells with EGF did not increase its binding. On the other hand, the starvation for 24hours decreased. We found that H522 lung adenocarcinoma cells overexpressed HER2 receptor, but less EGFR, compared with the other thoracic cancer cell lines, and that GEP100 endogenously bound HER2, neither EGFR nor HER3 in this cells. In vitro invasion of H522 cells was little promoted with EGF stimulation used as chemoattractants. It suggested that the signal transduction through GEP100 was mainly originated from HER2 signaling in H522 cells, not EGFR.

In vitro invasive activity in H522 cells was decreased by approximately 40-60% with three conditions as follows: the deprivation of HER2 signaling with the selective inhibitor, GEP100 knockdown by siRNA, or the interruption of this binding with the exogenous expression of PH domain of GEP100. And the knockdown of Arf6 and AMAP1, the downstream molecules of GEP100, similarly decreased in vitro invasion.

Immunohistochemical analyses of GEP100 and HER2 expression for 112 cases of resected specimens of human primary lung adenocarcinoma were performed and all samples were scored with the intensity of tumor lesion. These analyses showed that the intensity scores of GEP100 and HER2 were both statistically higher in pN2 cases than in pN1 cases. Furthermore, in cases of very strong HER2 expression (score; 3+), positive expression of GEP100 tended to be correlated with the high frequency of lymphatic metastasis (100% vs 61.5%, p=0.08).

GEP100 binding to HER2 receptor promotes lung cancer invasion and metastasis. Taking account of the importance of GEP100 in invasive mechanisms related with both EGFR and HER2 signaling, this GEP100 molecule appears to be a quite promising target to prevent cancer invasion and 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 5192.
Abstract 5196: Granulocyte-colony stimulating factor (G-CSF) plays a causative role for H-Ras-mediated invasion and migration of human breast epithelial cells
Authors: PARK SUN-HWA , MOON AREE
Published: February 2011

The whole human genome microarray on normal MCF10A, N-Ras-transformed, non-invasive MCF10A and H-Ras-transformed invasive MCF10A human breast epithelial cells revealed that the granulocyte-colony stimulating factor (G-CSF) was most highly upregulated in an H-Ras-specific manner. The present study investigated whether G-CSF plays a causative role for H-Ras-mediated MCF10A cell invasion and migration. Importantly, siRNA-mediated knockdown of G-CSF expression significantly reduced H-Ras-induced matrix metalloproteinase(MMP)-2 expression as well as invasion/migration. Activations of Rac1, MKK3/6 and p38 MAPK were inhibited by siRNA-knockdown of G-CSF. The induction of G-CSF expression conferred the invasive/migratory phenotype to parental MCF10A cells. Expression of MMP-2 and activation of signaling molecules Rac1, MKK3/6 and p38 MAPK were increased by G-CSF induction. Taken together, this study revealed G-CSF gene as a candidate marker for metastatic potential of breast epithelial cells.

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 5196.
Abstract 5197: Comparative proteome analysis reveals flotillin-1 as an H-Ras-interacting lipid raft protein critical for breast epithelial cell invasion
Authors: Yong Hae-Young , Hwang Jin-Sun , Kim Myeong-Ok , Sohn Hwa-Jin , Kim Eun-Sook , Choi Wahn Soo , Noh Dong-Young , Hwang Ki-Tae , Kim Hyeong-Reh Choi , Kim Ki-Bum , Lee Jae-Seon , Moon Aree
Published: February 2011

Metastatic spread is the major cause of death from breast cancer. The objective of our study is to unveil signal transduction pathways specific to breast epithelial cell invasion and migration apart from cell proliferation/transformation. We previously showed that the active H-Ras, but not N-Ras, induces invasive/migratory phenotypes of MCF10A human breast epithelial cells via activation of Rac1/p38 signaling leading to MMP-2 up-regulation, while both H-Ras and N-Ras induce cell proliferation and transformation. In the present study, we hypothesized that the Ras isoform-specific signaling pathway is initiated by differential involvement of lipid raft proteins. To address this, we characterized proteome profiles of lipid raft proteins from three cell lines: normal MCF10A; N-Ras-transformed, non-invasive MCF10A; and H-Ras-transformed, invasive MCF10A cells. Among twenty-four lipid raft proteins that were up-regulated in an H-Ras-specific manner, we tested whether flotillin-1 plays a causative role for H-Ras-mediated MCF10A cell invasion/migration. Here, we show that flotillin-1 interacts with H-Ras and H-Ras signaling increases flotillin-1 expression, suggesting a signaling amplification loop between flotillin-1 and H-Ras. Importantly, siRNA-mediated flotillin-1 knockdown significantly reduced H-Ras-mediated motility/invasion accompanied with down-regulation of Rac1/p38 and MMP-2. We also show that flotillin-1 co-localizes with H-Ras in the plasma membrane, while the interaction between flotillin-1 and N-Ras was negligible. Taken together, this study identified flotillin-1 as an H-Ras-interacting lipid raft protein critical for activation of H-Ras-specific signaling resulting invasive phenotype in breast epithelial cells. Consistently, flotillin-1 mRNA was significantly higher in malignant breast cancer tissues, supporting our hypothesis that flotillin-1 may contribute to breast carcinoma invasion.

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 5197.
Abstract 52: Overexpression of uridine phosphorylase 2 (UPP2) in tissues of uridine phosphorylase (UPP1) knock-out mice
Authors: Gao Yang , Ziemba Amy , Turner Jennifer , Pizzorno Giuseppe
Published: February 2011

Uridine phosphorylase (UPase) and thymidine phosphorylase (TPase) play critical roles in the metabolism of pyrimidines and their analogs used in cancer chemotherapy. These enzymes catalyze the reversible conversion of uridine, deoxyuridine and thymidine to the corresponding free bases and ribose or deoxyribose-1-phosphate. Uridine phosphorylase activity, in particular, has been reported to be elevated in breast cancers, colorectal cancer, oral squamous carcinomas, melanoma tissue, and lung adenocarcinomas when compared with adjacent normal tissues. Recently, a novel pyrimidine phosphorylase has been identified, called UPase-2. In human tissues, UPase-2 is predominantly expressed in kidney tissue, while in mouse tissue it is predominately expressed in the liver. Our recent studies have indicated that the UPase-2 isoform is overexpressed in UPase-1 knockout mice we have recently developed. A 37kDa (predicted MW of UPase-2) protein is overrepresented in liver lysate of UPase-/- mice after western blot analysis using a uridine phosphorylase polyclonal antibody. Real time RT-PCR analysis using UPase-2 specific primers shows a 4-fold increase in liver UPase-2 expression in UPase-/- mice compared to wild type mice. We also observed an increase in the expression of this enzyme in kidney and brain tissues; however, the enzymatic activity was below the limit of detection. No detectable mRNA levels were observed in spleen, lung or heart tissues. Interestingly, UPase-2 mRNA expression in wild-type and UPase-/- liver tissue decreased to 32% and 8% respectively after mice were fed diets containing 0.5% fenofibrate for 15 hours, however, UPase-1 mRNA expression increased 3.8-fold in the livers of wild-type mice. Fenofibrate, a ligand for peroxisome proliferator-activated receptor alpha, is used to treat high cholesterol and triglyceride levels, calling into question the role of UPase in lipid metabolism. Both the physiological role of UPase-2 in nucleoside metabolism and the homeostatic regulation of UPase-1 and UPase-2 in liver are presently unclear. To address this question, we plan to study this newly developed animal model to characterize UPase-2 contribution to pyrimidine and fluoropyrimidine metabolism, and the regulatory mechanisms affecting its expression and function.

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 52.
Abstract 521: Impaired migration of human fibrosarcoma cells HT1080 on glycated collagen type I
Authors: Said Georges , Guilbert Marie , Millerot-Serrurot Emilie , Schneider Laurence , Terryn Christine , Garnotel Roselyne , Jeannesson Pierre
Published: February 2011

Although a strict link does exist between cancer and aging, elderly patients present less invasive tumors than their younger counterparts. During aging, the fibrillar collagen type I, which is an abundant component of the extracellular matrix, becomes a prominent target of post-translational modifications such as glycation. Our present study is mainly designed to investigate the effect of non-enzymatic glycation of this long-lived protein on the proliferation and migration of human fibrosarcoma cells HT1080. For this purpose, collagen type I extracted from rat tail tendons was used for in vitro glycoxydation with ribose. Tumor cell behaviour was then evaluated on 2-dimensional plastic surfaces coated with native or glycated collagen type I. Results showed that the proliferation rate of HT1080 cells on glycated collagen type I did not differ from that on native collagen I after 1, 2 and 3 days of culture. Furthermore, the migration ability of HT1080 cells was studied by quantifying cell speed and trajectories via time-lapse videomicroscopy. At day 1, glycated collagen type I distinctly slowed down the cell locomotion by decreasing the mean cell speed by 47% (20.2 μm/h for glycated versus 38.3 μm/h for its native form). Continuous single cell tracking also permitted the investigation of cell trajectories. Cells grown on glycated collagen exhibited shorter or circular trajectories around their starting point in accordance with the cell speed inhibition. Because actin and vinculin appeared as key proteins involved in cell locomotion, we next investigated the effect of glycated collagen on the organization of these proteins in individual cells in motion. Cells exhibited a dramatic disorganization of F-actin cytoskeleton with loss of stress fibers as compared to native collagen. Actin was principally localized at the rim of the cell. Disturbance of actin integrity was accompanied with a disorganization of vinculin that also localized at the cell periphery. The impact of glycated collagen on the non-receptor protein tyrosine kinase FAK, which plays a crucial role in focal adhesion formation and cell locomotion, was also evaluated. We found that at day 1, glycated collagen significantly inhibits FAK expression by 39% but did not affect their phosphorylation state. These data clearly suggest that age-related changes of extracellular matrix proteins may contribute to a less invasive behaviour of tumor cells.

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 521.
Abstract 523: Three-dimensional hyaluronic acid-based hydrogel systems for studies of cancer invasion
Authors: Gurski Lisa A. , Nguyen Ngoc T. , van Golen Kenneth C. , Jia Xinqiao , Farach-Carson Mary C.
Published: February 2011

Currently available invasion assays for study of cancer metastasis have limitations that led us to develop a novel system that provides a more flexible, quantifiable, and physiologically relevant method to study cancer cell invasion. The three-dimensional (3D) hyaluronic acid (HA) hydrogel invasion assay then was used to study HA receptor signaling as it occurs during prostate cancer metastasis to bone marrow. Our invasion assay uses the HA hydrogel which reflects the prevalence of HA in the bone marrow and its key role in the promotion of cancer cell motility and tissue invasion. The HA hydrogel spontaneously crosslinks via interaction of aldehyde and hydrazide groups, allowing for direct encapsulation of cells within the gel. This cell/gel construct is plated in a 12 mm tissue culture insert for construction of the invasion assay. The cells are situated on a single plane so that individual cell movement from that plane can be tracked. We show that our 3D HA hydrogel invasion assay allows for cell migration/invasion in response to a chemoattractant, fetal bovine serum (FBS), and that this movement is easily quantified using confocal microscopy techniques over a distance of up to 1 mm. The ability of cancer cells to invade the HA hydrogel depends on their expression of HA receptors and hyaluronidases; more highly invasive cell lines express HA receptors and hyaluronidases at higher levels. We will show that culture in the HA hydrogel triggers differential expression and/or compartmental translocation of HA receptor proteins and downstream effectors, all of which contribute to motility and metastasis. Ongoing work examines the role that a specific HA receptor, RHAMM, plays in prostate cancer metastasis, specifically how it activates Rho GTPases that increase cell motility. We will show how these physiologically relevant 3-D HA hydrogel invasion assays can be used to study such pathways.

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 523.
Abstract 5232: Phosphatidylethanolamine is a marker of tumor vasculature and can be used as a target for optical imaging
Authors: Stafford Jason H. , Li Shuzhen , Thorpe Philip E.
Published: February 2011

New imaging agents are needed that home rapidly to tumors and saturate their target molecules with the unbound residue then clearing rapidly from the blood stream. Such agents should enable rapid detection of tumors. We have found that a phosphatidylethanolamine-binding peptide known as duramycin is an effective targeting moiety for construction of tumor-specific imaging agents. Phosphatidylethanolamine (PE) is one of two major phospholipids that present in the inner leaflet of the cell membranes, the other being phosphatidylserine (PS). We have previously demonstrated that oxidative stress conditions within the tumor microenvironment cause PS to flip to the outer leaflet in tumor endothelial cells. The exposed PS provides a highly specific marker of tumor vasculature. Since PE and PS distribution within the membrane is co-regulated, we hypothesized that PE would also be a marker of tumor vasculature. To target exposed PE, we generated several biotinylated duramycin constructs. Biotinylated duramycin bound to PE which became exposed on endothelial cells after they had been subjected to oxidative stresses. When the biotinylated duramycin was injected intravenously into mice bearing different types of tumors it rapidly localized to tumor vasculature and did not bind blood vessels in normal tissues. Next, we conjugated duramycin conjugated to a near-infrared fluophore (IRDye 800CW) and used it for optical imaging of subcutaneous RM-1 prostate tumors in mice. The duramycin-targeted near infrared probe efficiently labeled tumors and did not accumulate in non-target tissues giving clear detection of the tumors. These results show that PE exposure, like PS exposure, is a marker of tumor vasculature in many types of cancer. Duramycin labeled with detectable probes may have broad application in cancer imaging.

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 5232.
Abstract 5234: Sequential multi-modality imaging to detect metabolic changes during tumor growth
Authors: Serganova Inna , Moroz Maxim , Rizwan Asif , Ni Xiaohui , Moroz Ekaterina , Koutcher Jason , Blasberg Ronald
Published: February 2011

The expression and activity of lactate dehydrogenase A (LDH-A) in malignant tissue is considered a prognostic marker of poor outcome in many tumors. A limitation of tissue LDH-A measurements is the requirement of tissue for assay. The development of new non-invasive methods is highly demanded for clinical evaluation. One mechanistic link between tumor cell metabolism and the activity of lactate dehydrogenase A (LDH-A) is based on the HIF-1 transcription factor and its upregulation in tumors. HIF-1 binds to the LDH-A promoter and increases LDH-A expression; this in turn results in increased lactic acid production. The objective of this study was to detect the metabolic changes during tumor growth using a multi-modality imaging approach. To monitor HIF-1 pathway activity by bioluminescence imaging (BLI), we cloned an external Gaussia luciferase (exGLuc) reporter gene into an -IRES-GFP cassette, and placed the cassette in a self-inactivated retroviral vector under the control of an enhancer, containing multiple copies of the binding sites for the HIF-1 transcription factor. 18F-fluorodeoxyglucose (FDG)/microPET imaging was used for the detection of glucose metabolism in tumors, while lactate production was measured by proton magnetic resonance spectroscopic imaging (MRI/S). To validate this multi-modality imaging approach, human glioblastoma U87MG cells were transduced with the HIF-1 reporter vector and the HIF-1 reporter response was validated in vitro by exposure cells to hypoxia (1% O2) and to CoCl2 or DFO (deferoxamine mesylate). Despite a high basal level of HIF-1 transcriptional activity in the U87MG reporter cells, a strong correlation between oxygen deprivation and the level of exGLuc/GFP expression (indicating HIF-1 transcriptional activation) was detected, and this increase corresponded with a significant increase in LDH-A expression, assessed by immunoblotting. In vivo experiments using U87 reporter-xenografts also demonstrated a correspondence between the levels of HIF-1 expression and lactate production assessed by MRI/S. The measurement of lactate production using MRI/S with consecutive BLI imaging of HIF-1 activity and 18F fluorodeoxyglucose (FDG)/microPET confirmed the feasibility of a multi-modality imaging approach to study the link between the HIF-1 activity and a lactate production, as well as the level of glucose (FDG) uptake in growing xenografts. This multi-modal imaging paradigm can provide new and relevant information, and the opportunity to initiate and monitor more effective treatment strategies to arrest tumor growth.

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 5234.
Abstract 5239: Relationship between metabolite content on magnetic resonance spectroscopy and contrast enhancing fraction on MRI in patients with astrocytoma
Authors: McLean Mary , Sun Amy , Bradstreet Thomas , Schaeffer Andrea K. , Liu Haiying , Blenman-Abage Rose-Ann , Herman Gary , Price Stephen , Griffiths John
Published: February 2011

Background: Both magnetic resonance spectroscopy (MRS) and contrast-enhanced MRI are useful diagnostic tools for astrocytoma, but two important MRS peaks - lactate and lipid - are usually not resolvable. We investigated the relationship in astrocytoma between metabolite content measured in vivo using an MRS method that separated lactate from lipid, and tumor aggressiveness as reflected by contrast enhancing fraction (CEF) on MRI.

Method: Nine patients with astrocytoma (7 Grade IV, 2 Grade III) had both MRS and contrast-enhanced MRI. For MRS, spectra were edited to obtain separate estimates of lactate and lipid1 as well as N-acetyl aspartate (NAA), creatine, and choline from one 8 cm3 volume of interest (VOI) prescribed within the tumor boundary, avoiding areas of visible necrosis or cysts, and one matching VOI in the contralateral hemisphere. The CEF within the VOI was determined by thresholding T1-weighted images following Gadobutrol administration.

Results: Consistent with previous reports2, metabolite content differed markedly in tumor compared to contralateral tissue. Tumors contained higher levels of lactate and lipids and lower NAA and creatine. Choline did not differ significantly between tumor and contralateral tissue. The average CEF was 0.27, with a range from 0.0 in a Grade III tumor to 0.53 in a Grade IV tumor (that is, 53% of the pixels within the Grade IV VOI showed bright signal due to contrast uptake). The small size of the study precluded a formal subgroup analysis by histological grade, but qualitative observation suggests that the CEF, lipid, and lactate were all higher in tumors of Grade IV than III, consistent with expectations. The level of lipid showed a significant positive correlation with CEF (r=0.67, 95% CI 0.01, 0.92), while the numbers were too small to confirm any significant trends for the other metabolites.

Conclusion: In this first study to discriminate lactate from lipid and also compare MRS patterns with contrast enhancement in the same tissue volume, we found a positive correlation between lipid signal and CEF. Lipids, lactate, and CEF all appeared higher in higher-grade tumors on qualitative assessment and all merit further investigation as potential biomarkers of tumor aggressiveness.

References: 1Star-Lack J et al, J. Magn. Reson. 1998; 133:243-254. 2Howe FA et al, Magn. Reson. Med. 2003; 49:223-232.

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 5239.
Abstract 524: Interaction of metastatic breast cancer cells with osteoblasts and osteoclasts in a 3D in-vitro model
Authors: Krishnan Venkatesh , Vogler Erwin A. , Mastro Andrea M.
Published: February 2011

Breast cancer frequently metastasizes to bone where it causes osteolytic lesions. Although, the bone osteoclasts, not the cancer cells themselves, appear to be responsible for the bone resorbtion, we have evidence from 3D in vitro cell culture studies (bioreactor) that in the presence of breast cancer cells or their medium, the osteoblasts no longer make the proteins required for bone repair. Furthermore, they produce inflammatory cytokines, which in turn can stimulate osteoclasts.

We have developed a bioreactor system that permits continuous growth of bone-accreting osteoblasts (MC3T3-E1) for > 10 months without subculture or perfusion. This results in a mineralizing, multiple-cell-layer tissue that strongly resembles normal bone. We have also shown that, by challenging this osteoblast tissue with human metastatic breast cancer cells (MDA-MB-231GFP), important hallmarks of cancer metastasis including single cell filing of cancer cells, cancer-cell penetration of tissue and colonization can be observed. We hypothesize that addition of osteoclast to this 3D mineralizing osteoblast in the bioreactor would be a relevant in vitro bone surrogate for studying stages of breast-cancer metastasis. In standard tissue culture and in the bioreactor, we have been able to differentiate osteoclasts from hematopoietic progenitor cells isolated from murine bone marrow. The osteoclasts formed by exposure to RANKL and M-CSF were multi-nucleated, TRAP positive cells. The pre-osteoclast cells when introduced onto the osteoblast grown in the bioreactor, differentiated to form multinucleated resorbing osteoclast, also positive for TRAP and actin ring staining. To further improve the bone remodeling aspect, pre-osteoblasts will be re-infused into the system and allowed to ‘re-mineralize’ the matrix resorbed by osteoclasts. Once the system is established, breast cancer cells will be added to examine the three way cell interactions. This 3D in-vitro cell culture system containing both osteoblast and osteoclast will help to further understand the cellular and molecular events that lead to successful breast cancer colonization of bone. Positive outcomes of the work will help clarify the etiology of metastasis but also create a much-needed in vitro tool for study of cancer therapeutics.

This work was supported by US Army Medical and Materiel Command Breast Cancer Idea Program (W81XWH-06-1-0432).

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 524.
Abstract 5245: Regional analysis of DCE MR images to unravel tumor growth and response heterogeneity in SW-620 human colon adenocarcinoma xenografts
Authors: Kaimal Vinod , Meade MaryAnne , Woolliscroft Michael , McConville Patrick
Published: February 2011

Introduction: Dynamic Contrast-Enhanced (DCE) MRI, a clinically relevant technique used to assess response to anti-angiogenic therapies, relies on imaging of a T1 modifying contrast agent to characterize vascular response by the dynamics of tracer exchange between the vessels and the interstitial space. The parameter Ktrans is a measure of the tumor vascular permeability, vascular surface area and blood flow, and is normally assessed as mean over the entire tumor volume. Generally, decreasing tumor mean Ktrans occurs after effective vascular targeting. However, localized tumor growth based necrosis/under-perfusion can also lower Ktrans, leading to uncoupling of the endpoint from the effect. Techniques that can overcome confounding effects of heterogeneity may improve Ktrans sensitivity and correlation with outcome. We applied a thresholding technique to Ktrans data to analyze regional Ktrans variations in subcutaneous SW-620 tumors treated with the vascular targeted agent, bevacizumab or vehicle. Materials and Methods: Female nu/nu mice were implanted with SW-620 tumor fragments on Day 0. On Day 14, mice were distributed into groups with matching tumor burden. DCE-MRI images were acquired 24 hours before treatment (vehicle or bevacizumab; IP; 5mg/kg; Q3Dx4), and one week after treatment. The data were fitted to a Tofts-Kermode kinetic model to generate Ktrans over the entire tumor volume. Thresholds we determined by pooling all the Ktransvalues from the pre-treatment DCE MRI scans. The Ktrans values for each image were distributed into low, mid and high Ktrans fractions and the percentage of voxels in each fraction was calculated. Results: Bevacusimab resulted in moderate tumor growth inhibition (T/C = 43% on Day 22). Tumor mean Ktrans one week post-treatment start did not correlate with this effect, with Ktrans (T/C) = 131%. Analysis of location of voxels in the low, mid and high Ktrans fractions in the pre-treatment images showed that lower Ktrans values were generally confined to the tumor core, while higher Ktrans values were generally confined to the tumor periphery. These regions presumably related to under-perfused (core) and well perfused (periphery) tissue. A comparison of the high Ktrans volume fractions one week post-treatment (treated vs control) showed a correlation with the growth based treatment effect (high Ktrans volume fraction (T/C) = 44%). Ktrans thresholding may therefore provide a means to increasing sensitivity to treatment-based vascular inhibition.

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 5245.
Abstract 5248: Neuroblastoma tumors with different ALK mutations are sensitive to Perifosine
Authors: Li Zhijie , Thiele Carol J.
Published: February 2011

Background: Neuroblastoma (NB) is the most common extracranial solid tumor of children. Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase predominantly expressed in the developing nervous system. Recently, mutated ALK has been identified in association with familial and sporadic neuroblastoma (NB). Patients whose tumors contain amplification, mutation or express increased levels of activated ALK have a poor prognosis. Downstream targets of ALK include the STAT and Akt pathways. Perifoisine is an Akt inhibitor that has been reported to inhibit tumor growth in adult pre-clinical models. The aim of this study was to assess whether ALK mutations affect the response of NB cells to perifosine.

Methods: We selected 4 NB cell lines with different ALK mutation status (SY5Y-ALK F1174L mutation; KCNR-ALK R1275Q mutation, AS and NGP with wild-type (wt) ALK. The effect of perifosine on Akt phosphorylation and cell survival was evaluated in these cell lines. In vivo studies were also performed in nude mice by subcutaneous injection of NB cells with or without ALK mutation. The tumor growth and phosphorylated Akt levels in tumor tissues were studied.

Results: In vitro, treatment with perifosine induced decreases in Akt phosphorylation in all the 4 cell lines. SY5Y-ALK F1174L which is reported to be sensitive to ALK inhibitor TAE684, and KCNR-ALK R1275Q, AS-ALK wt and NGP-ALK wt which are reported to be resistant to ALK inhibitor TAE684, all responded to perifosine treatment. In vivo, perifosine treatment delayed or inhibited growth of tumors containing mutant or wild-type ALK. The phosphorylated Akt levels were lower in tumors treated with perifosine compared to tumors treated with control reagent.

Conclusions: These data indicate that Perifosine is able to control NB tumor cell growth despite the mutational status of ALK. Perifosine may be a candidate treatment for NB tumors not only for those with wt ALK but especially for those that carry mutations that are resistant to ALK inhibitor treatment.

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 5248.
Abstract 525: ADAM-9 expression in gastric cancer and its association with invasiveness
Authors: Kim Jeong Min , Yu Eun Jeong , Jung Jae Joon , Kim Tae Soo , Rha Sun Young , Ahn Joong Bae , Ahn Jung Ryun , Kim Chan , Chung Hyun Cheol , Roh Jae Kyung , Jeung Hei-Cheul
Published: February 2011

The ADAMs (a disintegrin and metalloproteinase) comprise a family of multidomain transmembrane and secreted proteins. Accumulating evidence associates an increased expression of individual ADAM family members with various types of cancer including prostate, breast, skin and lung cancers. However, little is known about its role in gastric cancer. Herein, we investigated the expression of ADAM-9 in gastric cancer, and its implication as a potential therapeutic target for anti-metastaasis therapy.

First, we evaluated the expression of ADAM-9 on protein by immunoblotting and transcript level in 24 gastric cancer cell lines by RT-PCR. ADAM-9 protease activity was also measured using ADAM-9 fluorogenic substrate (R&D systems, Minneapolis, MN, USA). We then conducted invasion assay using transwell coated with matrigel in the presence or absence of ADAM-9-specific inhibiting antibody (kindly supplied from Raven Biotechnologies, Inc., South San Francisco, CA). The mRNA expression of ADAM-9 was observed in all the gastric cancer cell lines while the protein expressions of pro- and active-form and protease activities were variable. We selected the cell lines of high baseline ADAM-9 expression (SNU-638, MKN-28 and AGS) and of low baseline expression (SNU-216, MKN-1 and HS-746T) to continue further experiment. Baseline level of ADAM-9 is little correlated with invasion activity though matrigel. Treatment of anti-ADAM-9 antibody does not influence the cytotoxicity. However, it reduced its protease activity and invasiveness, which was dependent on baseline ADAM-9 activity. Actually, protease activity reduced 64.5±0.3% when the specific antibody to ADAM-9 was treated to high expressed cell lines, but 9.6±0.4% were decreased in low expressed cell lines at the 20ug/ml of the antibody. Anti-ADAM-9 antibody inhibited the invasion activity dose dependently, and its effect was more prominent in high ADAM-9-expressing cell lines (67.7±1.9% vs 5.9±4.3% at the 20 ug/ml antibody). Anti-ADAM-9 treatment did not influence on the cell-matrix adhesion, which implies this anti-invasive effect is through its inhibition of protease function. In the hypoxic condition (1% oxygen), invasion was increased along with elevation of both active form of ADAM-9 and its protease activity, suggesting that increased ADAM-9 could have a role of enhancing invasiveness of gastric cancer. To conclude, we presented that ADAM-9 has a role in invasiveness of gastric cancer and would be one of the putative targets for anti-metastasis therapy.

This work was supported by the Brain Korea 21 Project for Medical Science, Yonsei University.

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 525.
Abstract 5273: The cyclin dependent kinase inhibitor p27KIP1 inhibits neuroblastoma cell migration and invasion
Authors: Koomoa-Lange Dana-Lynn T. , Bachhmann Andre S.
Published: February 2011

Neuroblastoma (NB) is the most common extra-cranial pediatric tumor. Over 70% of NB patients are diagnosed with metastasis, and the survival probability of these patients is significantly lower than patients without metastasis. While the role of p27KIP1 in cell cycle regulation is well documented and conserved in all cell types, there are contradictory reports regarding its function in regulating cell migration and invasion. The purpose of the present study was to elucidate the role of p27KIP1 in regulating migration and invasion of NB cells with MYCN over-expression. Western blot analysis, migration and invasion assays were performed on NB cells with and without over-expression of MYCN. The expression of p27KIP1 was down-regulated using siRNA, and the migration rate was determined on NB cells with MYCN over-expression. Also, the cytoplasmic localization of p27KIP1 in NB cells with and without MYCN over-expression was determined. The results from the current study show that MYCN over-expression increases migration and invasion of NB cells, compared to cells without MYCN over-expression via a process that involves p27KIP1. In addition to its role as a cell cycle regulator, p27KIP1 also regulates NB migration and invasion. This dual role of p27KIP1 as a tumor repressor suggests that it may be an attractive therapeutic target for NB.

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 5273.
Abstract 5274: Role of Bcl-XL and STAT3 in γ-irradiation-induced invasion of lung cancer cells
Authors: Ho Jin-Nyoung , Kang Ga Young , Um Hong Duck
Published: February 2011

Previous reports suggest that, besides its therapeutic effects, ionizing radiation (IR) may increase the invasiveness of surviving cancer cells. Here, we show that this activity of IR in lung cancer cells is mediated by a signaling pathway that involves p38 kinase, phosphoinositide 3-kinase, Akt, and MMP-2. The invasion-promoting doses of IR also increased and reduced the levels of vimentin and E-cadherin, respectively, both of which are markers for the epithelial-mesenchymal transition (EMT). Interestingly, all these actions of IR were mimicked by the overexpression of Bcl-XL in lung cancer cells. Moreover, both RNA and protein levels of Bcl-XL were elevated upon irradiation of the cells, and the prevention of this event using small interfering RNAs of Bcl-XL reduced the ability of IR to promote invasion signals and EMT-associated events. This suggests that Bcl-XL functions as a signaling mediator of the malignant effects of IR. It was also demonstrated that IR enhances levels of STAT3 phosphorylation, and the reduction of STAT3 levels by RNA interference prevented IR-induced Bcl-XL accumulation, and thus all the tested Bcl-XL-dependent events. Overall, the data suggest that IR induces Bcl-XL accumulation via STAT3, which then promotes cancer cell invasion and EMT-associated markers. Our findings demonstrate a novel function of Bcl-XL in cancer, and also advance our understanding of the malignant actions of IR significantly.

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 5274.
Abstract 5280: Exchange protein activated by cAMP (Epac) accelerates melanoma cell migration through Ca 2+-dependent actin assembly
Authors: Baljinnyam Erdene , Nowycky Martha , Iwatsubo Kousaku
Published: February 2011

Background: We previously reported that Epac, a guanine nucleotide exchange factor directly activated by cAMP, increases melanoma cell migration through Ca2+ elevation. Both Epac-induced cell migration and Ca2+ elevation were reduced by pharmacological inhibition of phospholipase C (PLC) and inositol triphosphate (IP3) receptor. Accordingly, we studied the involvement of PLC/IP3 receptor in Epac-induced Ca2+ elevation by knockdown of these molecules. However, the mechanism how Epac-induced Ca2+ elevation mediates melanoma cell migration is unknown. Here, we investigate the effect of Epac induced-Ca2+ elevation on actin assembly, which plays a major role in cell migration.

Methods: SK-Mel-2, a cell line from human metastatic melanoma, was used in this study. Intracellular Ca2+ was measured with Fura-2AM fluorescent dye. Cell migration was examined with the Boyden chambers. IP3 production was examined by IP3 radioimmunoassay. Actin assembly was examined by immunocytochemistry. In some experiments, ablation of a target protein with lentivirus-based shRNA induction system was performed.

Results: 8-pMeOPT-2′-O-Me-cAMP (8-pMeOPT), an Epac-specific agonist, increased Ca2+ and cell migration in SK-Mel-2. Such Ca2+ elevation and cell migration was negated by ablation of PLCε or IP3 receptor 1. In addition, Epac-induced IP3 production was inhibited by U73122, a PLC inhibitor, and ablation of PLCε. These data suggest that Epac increases Ca2+, and thus cell migration, via the PLCε/IP3 receptor 1 signaling pathway. Cytochalasin D, an actin assembly inhibitor, reduced 8-pMeOPT-induced cell migration. In addition, immunocytochemistry showed that 8-pMeOPT increases actin assembly. When U73122 or Xestopongin C, an IP3 receptor inhibitor, was used, Epac-induced actin assembly was decreased. These data suggest that Epac-induced Ca2+ elevation mediates actin assembly. Further, immunoprecipitation showed that 8-pMeOPT increased physical interaction of myosin heavy chain IIA (MHCIIA), which enhances actin assembly, with S100A4, a Ca2+-binding protein which activates MHCIIA. These data suggest that Epac-induced Ca2+ elevation mediates actin assembly via the S100A4/MHCIIA signaling pathway.

Conclusion: Epac increases Ca2+ via the PLCε/IP3 receptor 1 pathway. Also, Epac-induced Ca2+ elevation mediates melanoma cell migration via actin assembly. These data suggested that Epac could be a target for treating patients with melanoma.

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 5280.
Abstract 5286: Beta-arrestin2-CARMA3 signaling axis is required in lysophosphatidic acid-induced ovarian cancer migration and invasion
Authors: Sun Jiyuan , Cai Jingli , Feng Yumei , Guo Lei
Published: February 2011

Ovarian cancer is ranked as the 4th most common type of cancer among women in the United States. One of the biomarkers of ovarian cancer is lysophosphatidic acid (LPA). LPA is a type of G protein-coupled receptor ligand. It is strikingly elevated in 90% of ovarian cancer patients. Additionally, LPA receptors are aberrantly up-regulated in ovarian cancer patients. We have reported previously that a signaling axis consisting of beta-arrestin 2 and CARMA3 (CARD and MAGUK domain-containing protein 3) is indispensable in LPA-induced nuclear factor kappa B (NF-kappaB) activation in mouse embryonic fibroblast cells. NF-kappaB is an important transcription factor, which has been suggested to mediate LPA-induced ovarian cancer migration and invasion. However, it remains unknown whether the beta-arrestin2-CARMA3 signaling axis plays an important role in LPA-induced ovarian cancer cell migration and invasion. In the present study, using beta-arrestin 2 and CARMA3 shRNA, we knockdowned their protein expression levels in ovarian cancer cell lines. Consistent with previous reports, we found that down-regulation of beta-arrestin 2 and CARMA3 abolished LPA-induced IKK activity and NF-kappaB activation in ovarian cancer cells. In addition, in vitro transwell migration and matrigel invasion assays demonstrated that beta-arrestin 2 and CARMA3 shRNA significantly impaired LPA-induced ovarian cancer cell motility and invasiveness. Together, our results provide the evidence that beta-arrestin 2 and CARMA3 serve as critical regulators in LPA-induced, NF-kappaB-mediated ovarian cancer migration and invasion. Therefore, we speculate that beta-arrestin 2 and CARMA3 may represent attractive therapeutic targets for ovarian cancer and many other types of malignancies.

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 5286.