bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5190: GnRH promotes peritoneal adhesion and dissemination of ovarian cancer cells
Authors: Cheung Lydia W. T. , Leung Peter C. K. , Wong Alice S. T.
Published: February 2011

Ovarian cancer has the highest mortality of all gynecological cancers. It is often diagnosed at a late stage characterized by widespread peritoneal dissemination and malignant ascites. A key initial step in metastasis is the adhesion of ovarian cancer cells to the peritoneal mesothelium and extracellular matrix (ECM). Gonadotropin-releasing hormone (GnRH) receptor is present in 80% of ovarian carcinomas and its role in ovarian cancer cell migration/invasion has recently been revealed. Here, we show for the first time a role for GnRH in promoting ovarian cancer cell adhesion to the peritoneum. We observed that GnRH agonist (GnRHa)-stimulated cells were able to adhere to the peritoneal mesothelium efficiently. Moreover, the expression of integrins (α2, α5 and β1) in ECM recognition for collagen I, laminin, and fibronectin were upregulated and activated by GnRHa. The adhesion of ovarian cancer cells to the mesothelium/ECM components activated by GnRHa could be blocked by neutralizing antibodies to α2, α5 and β1 integrin. Knockdown of the GnRH receptor using small interfering RNA significantly abrogated the GnRH-induced cell adhesion and integrin expression, confirming that these effects were GnRH receptor specific. Furthermore, we showed that downregulation of β1 integrin and GnRH receptor with short hairpin RNA-mediated gene silencing significantly reduced ascites formation and intra-abdominal metastasis in intraperitoneal xenografts of human ovarian cancer in nude mice. These results indicate that GnRH may play a critical role in the peritoneal adhesion and dissemination of ovarian carcinomas and could be a promising target in ovarian cancer (This work is supported by the Hong Kong Research Grants Council Grant HKU778108 to A.S.T.W.).

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 5190.
Abstract 5195: Shh/Gli signaling pathway promotes tumor metastasis and lymphangiogenesis via activation of PI3K/Akt and Erk pathway in gastric cancer
Authors: Yoo Young-A , Kang Myoung-Hee , Kang Han-Na , Kim Jung-Lim , Seo Jae-Hong , Kim Jun-Suk , Oh Sang-Cheul
Published: February 2011

The activation of Sonic hedgehog (Shh)/Gli signaling is involved in the progression and invasion of various tumors types, including gastric carcinoma. Epithelial-mesenchymal transition (EMT) and matrix metalloproteinases (MMPs) have been implicated in facilitating the invasion and metastatsis. Herein, we investigated the impact of phosphoinositide 3-kinase (PI3K)/Akt and Erk pathway and MMPs activity on the Shh/Gli-mediated EMT and invasion of gastric cancer cells. We found that overexpression of Shh significantly enhanced cellular motility/invasiveness, lymphangiogenesis, and metastatic ability of tumor cells in gastric cancer cell lines and mice. Stimulation of Shh in gastric cancer cells also induced a full EMT process (characterized by Snail induction and E-cadherin down-regulation) and expression and activity of MMP-9. Meanwhile, blockade of Shh/Gli signaling by Cyclopamine (a Shh signaling inhibitor), anti-Shh neutralizing antibodies, or Gli siRNA inhibited Shh-induced motility/invasiveness and lymphangiogenesis in gastric cancer by subsequently restoring these changes of EMT markers and activity of MMP-9. The phosphorylation of Akt and Erk was also enhanced by treatment with Shh, but not cyclopamine, anti-Shh neutralizing antibodies, or Gli siRNA. Blockade of the Akt and MEK kinase using kinase-dead Akt, LY294002, or PD98059 significantly inhibited the Shh-induced EMT, activity of MMP-9, lymphangiogenesis, and metastasis in tumor cell lines and mice over-expressing Shh. Furthermore, knock-down of MMP-9 by its siRNA results in an decrease in invasiveness of gastric cancer cells treatment with Shh. Immunohistochemistry on gastric tumor biopsies showed that the levels of Gli, E-cadherin, MMP-9, D2-40 (lymphangiogenic factor), and phospho-Akt expression were enhanced in cases of metastatic gastric cancer than in cases of primary gastric cancer. Moreover, the strong correlation between Gli and E-cadherin, MMP-9, D2-40, phospho-Akt, or phospho-Erk expression was also observed in lymph node metastasis specimens. These data indicate that Shh/Gli signaling pathway promotes tumor metastasis in gastric cancer through activation of PI3K/Akt and Erk pathway followed by up-regulation of EMT, MMP-9, and lymphangiogenesis, which results in the stimulation of invasiveness 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 5195.
Abstract 5199: Promotion of prostate cancer cell migration and invasion via CXCR3 signaling
Authors: Wu Qian , Wells Alan
Published: February 2011

Tumor cells exist within, and cross-communicate with their local milieu that includes non-neoplastic cells using a variety of soluble chemokines. CXCR3-binding chemokines have recently been implicated in breast cancer metastasis, though inversely correlated with prostate tumor growth. There are two CXCR3 isoforms differing by a 5′ splice variant, that vary in their distribution, with CXCR3A found mainly on cells of the hematopoietic lineage and CXCR3B the predominant species on adherent cells. These isoforms vary in their predilection for the four common ligands, and the consequences of signaling through these different isoforms can be divergent. How these two isoforms inform tumorigenesis and invasion still remains unclear. Here, we examined the expression and function of the two different variants of CXCR3 in prostate cancer cells. In contrast to normal prostate epithelial cells (RWPE-1), in which CXCR3B was the primary splice variant, CXCR3A was highly expressed in two tested prostate cancer cell lines (DU-145 and PC-3). Two known ligands of CXCR3, CXCL10/IP10 and CXCL11/IP9/I-TAC, were down-regulated in these two prostate cancer cell lines, while the CXCR3B-selective ligand CXCL4/PF4 was overexpressed, allowing for autocrine stimulation. Instead of inhibiting cell migration as in RWPE-1 cells, CXCL4/PF4 and CXCL10/IP10 promoted cell motility and invasiveness in both DU-145 and PC-3 cells in vitro, which might be a result of PLCβ3 activation. Overexpression of CXCR3B in DU-145 cells slightly decreased cell chemoattractant movement and invasion. These data suggest that the aberrant expression of CXCR3A and down-regulation of CXCR3B may promote prostate tumor metastasis via stimulating cell migration. (Funded by a VA Merit Award)

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 5199.
Abstract 5200: Role of MLK3 in breast cancer cell migration and metastasis
Authors: Sondarva Gautam , Mehrotra Suneet , Mishra Rajakishore , Rangasamy Velusamy , Fareed Jawed , Rana Basabi , Rana Ajay
Published: February 2011

Effectively treating breast cancer metastasis is one of the biggest challenges in breast cancer therapy. The overall poor prognosis associated with cancer cell migration to distal organs underscores the critical need to understand the biology of the disease in order to develop more effective therapies. Members of protein kinase family have often been targeted for therapeutic purposes in different types of cancer, including breast cancer, and several protein kinase inhibitors are currently in clinical trials for specific types of cancer.

The members of Mixed Lineage Kinases (MLKs) are novel family of MAP3Ks and their physiological function is not known, however, we and others have shown that several of MLK family members activate Jun- N-terminal Kinases (JNKs) and MLK activation regulates survival in neuronal cells. Recently, we have seen that MLK3 is highly expressed in invasive breast cancer cell lines and in primary breast tumors, compared to the normal mammary tissue from same patient. Since MLK3 was initially identified from platelet cell line, CMK11-5, and association of venous thrombosis and occult cancer was recognized almost 140 years ago, we hypothesized that MLK3 or other MLKs are important in proteinase receptor-mediated signaling. Furthermore, the downstream effectors of proteinase receptors (PARs) that mediate the breast cancer cell growth, migration and metastasis to distal organs are currently unknown.

We examined the role of MLK3 in thrombin mediated signaling in a highly invasive breast cancer cell line, MDA-MB-231. Thrombin treatment of these cells activated MLK3 kinase in a time dependent manner. MLK3 was activated within 2 minutes of thrombin treatment and the activity peaked at 5 minutes, and was associated with consequent activation of its downstream target JNK. Furthermore, pretreatment of MDA-MB-231 with an MLK inhibitor, CEP-11004 completely blocked thrombin-induced JNK activation. Importantly, we also discovered that thrombin induces MLK-dependent migration in MDA-MB-231 cell line was blocked by MLKs inhibitor, CEP-11004.

These results collectively suggest that MLK3 is an important target of proteinase receptors-mediated migration/invasion/metastatic pathways, and help us to identify MLK family members’ inhibitors as viable drugs to treat metastatic breast cancer.

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 5200.
Abstract 5210: PRL-1 promotes cell metastatic processes and regulates filamentous actin dynamics
Authors: Nakashima Masanao , Lazo John S.
Published: February 2011

The phosphatases of regenerating liver (PRL) are a family of the protein tyrosine phosphatases that have been hypothesized to be involved in metastatic cancer. While the PRLs appear to regulate cancer cell migration, invasion and proliferation, we do not fully understand mechanistically how they control these processes. In the current study, we present new findings demonstrating that PRL-1 regulates filamentous actin dynamics, which could promote cell metastatic processes. Human A549 non-small cell lung cancer cells stably expressing wildtype PRL-1 exhibited a 60% increase in cell migration at 72 hours and a three-fold increase in cell invasion at 24 hours. Cells expressing catalytic mutants of PRL-1 (C104S and D72A) did not demonstrate increased cell migration and invasion, indicating that these phenotypic changes required the PRL-1 phosphatase activity. PRL-1 siRNA decreased PRL-1 protein levels by 50% and decreased cell migration by 25%. Cellular invasiveness is controlled by the cadherin-catenin complex and dynamic filamentous actin. We found wildtype PRL-1 interrupted E-cadherin and regulated Rho family kinase function, which have important roles in actin filament assembly and stabilization of focal adhesion. We also observed that expression of wildtype PRL-1 caused diffusely disassembled F-actin with reduced F-actin levels as compared to that seen in cells expressing the vector control or the catalytic mutant. We conclude the PRL-1 enhanced lung cancer cell migration and invasion. These results suggest PRL-1 could be a productive therapeutic target for cancer metastasis and support further efforts to identify its substrates.

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 5210.
Abstract 5212: Akt signaling enhances carcinoid cell secretion, migration, and invasion
Authors: Silva Scott R. , Li Jing , Bowen Kanika A. , Townsend Courtney M. , Evers B. M.
Published: February 2011

Carcinoid tumors are slow growing neuroendocrine tumors characterized by the secretion of peptides and biogenic amines such as serotonin (5-HT) and TGF-β. Although relatively indolent, carcinoid tumors of the GI tract can metastasize to the liver and release secretory products into the systemic circulation resulting in the carcinoid syndrome. PI3K/Akt signaling is increased in carcinoid tumors and is hypothesized to promote carcinoid progression. The purpose of our current study was to delineate the role of Akt signaling in carcinoid cell secretion, migration, and invasion using the BON cell line, derived from a human pancreatic carcinoid tumor. Methods. i.) BON cells were transfected with either non-targeting control shRNA (BON shControl) or shRNA to PTEN (BON shPTEN), the natural inhibitor of Akt activation. Conditioned media was collected from BON shControl and shPTEN cells and assessed for 5-HT and TGF-β by ELISA. Western blots were performed to assess changes in the expression of 5-HT and TGF-β. ii.) Scratch and Boyden chamber assays were performed to compare the migration and invasion, respectively, of BON shControl and shPTEN cells. The protein levels of the epithelial-to-mesenchymal transition (EMT) markers E-cadherin, a cell adhesion protein, and snail were assessed in both cell groups with western blot. Cells were injected into the pancreas of athymic nude mice and liver metastases were assessed via immunofluorescence. Results. i.) Knockdown of PTEN increased the secretion of 5-HT and TGF-β from BON cells. Compared to control, lysates from BON shPTEN cells displayed increased expression of tryptophan hydroxylase, the rate limiting enzyme of 5-HT biosynthesis, and TGF-β. ii.) Compared to BON shControl cells, BON shPTEN cells exhibited a 3.2- and a 1.6-fold increase in migration at 24 h and 48 h, respectively. At 24 h BON shPTEN cells displayed an 8-fold increase in invasion, while at 48 h a 2.6-fold increase in invasion was noted compared to BON shControl cells. Knockdown of PTEN in BON cells significantly decreased E-cadherin expression, whereas snail expression was increased. BON shPTEN and BON shControl cells formed liver metastases in 60% and 10% of mice, respectively. Conclusions. The Akt pathway enhances carcinoid cell secretion and metastatic potential by increasing expression and synthesis of secretory products and by altering expression of EMT markers, respectively. In addition to promoting metastasis, Akt signaling may also contribute to carcinoid syndrome. These results suggest that targeting Akt signaling may provide a dual strategy to decrease peptide secretion and inhibit carcinoid cell invasion.

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 5212.
Abstract 5220: Early prediction of tumor response to APO866 treatment with 3′-deoxy-3′-[18F]fluorothymidine positron emission tomography in human ovary cancer xenografts in mice
Authors: Jensen Mette Munk , Erichsen Kamille Dumong , Björkling Fredrik , Madsen Jacob , Højgaard Liselotte , Sehested Maxwell , Kjær Andreas
Published: February 2011

Introduction: APO866 is a new anti-tumor compound found to inhibit nicotinamide phosphoribosyltransferase (NAMPT), an enzyme involved in the biosynthesis of NAD. The compound has anti-tumor effect in several pre-clinical tumor models and is currently in several clinical phase II studies. 3′-deoxy-3′-[18F]fluorothymidine (FLT) is a tracer used to assess cell proliferation in vivo. The aim of the study was to evaluate if FLT positron emission tomography (PET) may be used to monitor early anti-proliferative effects of APO866.

Methods: In vivo uptake of FLT in human ovary cancer xenografts in mice (A2780) was studied at various time points after APO866 treatment (15 mg/kg ip twice daily for 7 days) was initiated. Baseline FLT scans were made before either APO866 (n=10 tumors) or vehicle (n=6 tumors) was injected and repeated after 24 hours (2 treatments), 48 hours (4 treatments) and 7 days (14 treatments). Tumor volume was followed with computed tomography (CT) during the experiment. FLT uptake was quantified using small animal PET/CT. One hour after iv injection of 10 MBq FLT, static PET scans were performed for 10 minutes and region of interests (ROIs) covering whole tumors were defined on PET/CT images for calculation of standard uptake values (SUV). Comparison within treatment groups was performed using paired t-tests and between control and APO866 groups with unpaired t-tests. All p-values were corrected for multiple comparisons using the Bonferroni algorithm. P<0.05 was considered significant.

Results: Tumors treated with APO866 had volumes that were 122% (24h), 129 % (48h) and 88% (day 7) relative to baseline volumes day 0. In the control group tumor volumes were 123% (24h), 150% (48h) and 318% (day 7) relative to baseline volumes day 0. Tumor volume between groups were significantly different at day 7 (P=0.006). APO866 treatment caused significant decrease in uptake of FLT at 24 hours (−23%; P<0.001), 48 hours (−29%; P<0.001) and day 7 (−25%; P=0.0013) after treatment start compared to baseline uptake. In the control group uptake of FLT did not change.

Conclusions: APO866 treatment caused a significant decrease in FLT uptake 24 and 48 hours after injection of first dose. The early reductions in tumor cell proliferation preceded decrease in tumor volume on day 7. Our data indicate that FLT PET is promising for early non-invasive assessment of APO866 anti-tumor effects.

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 5220.
Abstract 5223: Towards MYC targeted cancer therapy: Noninvasive molecular imaging of c-Myc signaling
Authors: Fan-Minogue Hua , Cao Zhongwei , Ramasamy Paulmurugan , Carmel Chan , Massound Tarik , Dean Felsher , Gambhir Sanjiv
Published: February 2011

MYC protein regulates a variety of human genes and is deregulated in most of human cancers. The deregulation of MYC is at multiple levels, from gene transcription to mRNA translation and protein activity. Activation of Myc protein is phosphorylation-mediated and has been found to be essential for development of many cancers. Here, we developed a bioluminescent sensor, using protein assisted split luciferase complementation system, to noninvasively detect the phosphorylation-mediated activation of the cytoplasmic Myc (c-Myc). In this sensor system, the c-Myc phosphorylation motif and the phosphorylation recognition domain of GSK3beta were fused with the two split luciferase fragments respectively. Phosphorylation induced c-Myc interaction with GSK3beta resulted in complementation of the two split luciferases and reconstitution of luciferase activity. Signal of the optimal sensor was specific for the c-Myc phosphorylation. It detected as low as 2-fold higher levels of c-Myc phosphorylation in cancer cells versus normal cells. It also detected the temporal regulation pattern of c-Myc phosphorylation upon serum stimulation in normal cells. MAP kinase inhibitor, PD98059 and U0126, and Atorvastatin (AT), induced dose dependent signal reduction in tumor cells, which was correlated with the decrease of endogenous c-Myc phosphorylation. In mouse tumor xenograft models, the sensor detected the inhibitory effects of AT well before there was a detectable difference in tumor size (P<0.05). For the first time, we developed a sensor that can noninvasively monitor phosphorylation mediated c-Myc activation. It may also facilitate drug development by allowing rapid readout of inhibition effect in high throughput drug screen for MYC-targeted cancer therapy.

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 5223.
Abstract 5236: Intermittent anti-angiogenic therapy with sunitinib in a murine xenograft model monitored by dynamic contrast enhanced ultra high frequency sonography
Authors: Hunnam Titaina C. Potdevin , Tuthill Theresa A. , Dorow Donna S. , Cullinane Carleen , McCarthy Timothy J. , Hicks Rodney J.
Published: February 2011

Sunitinib is an orally bio-available small-molecule inhibitor that targets multiple pro-angiogenic receptor tyrosine kinases including VEGFR, PDGFR, c-KIT, FLT3, CSF-1R and RET. Sunitinib treatment induces growth arrest or regression in a range of preclinical tumour models via its anti-tumour and anti-angiogenic effects. An intermittent 4 weeks on, 2 weeks off sunitinib dosing schedule is routinely used in the clinic to reduce pharmacologic toxicity of the compound. However, the effects of treatment withdrawal on tumour vasculature are currently poorly defined. The aim of this study was to use dynamic contrast enhanced ultra high frequency sonography (DCE-UHS) to investigate the effects of intermittent therapy on vascular function in a preclinical xenograft tumour model. Nude mice bearing A431 human squamous cell tumours (mean volume 296 +/− 19 mm3) were randomized to receive either vehicle or 80 mg/kg sunitinib p.o. daily following a treatment regimen of 6 days on, 7 days off, 7 days on (n = 4/group). During the 20 day regimen period, mean tumour volumes in the sunitinib treated group regressed by 42% at day 6 but increased by 38% during the off treatment period days 6 - 13. Upon resuming sunitinib therapy, tumour volumes decreased once again by 13% (days 13 - 20). Mean tumour volumes in the vehicle treated group reached 561+/−27 mm3 at day 6 becoming too large for DCE-UHS measurements and were thus removed from the remainder of the experiment. DCE-UHS was performed at baseline and during the treatment regimen at days 4, 6, 11, 13, 17 and 20. Measures of tumour perfusion including relative fractional blood volume (RFBV; percentage of tumour volume with blood flow) and mean refill rate (MRR, mean rate at which blood re-perfuses a defined tumour area) are calculated from the DCE-UHS data. During the treatment periods, mean RFBV in the treated animals decreased by 52% (p = 0.011; days 0-6) and 55% (p = 0.021; days 13-20) and MRR by 77% and 72% (p values not significant). However, during the intervening off treatment period tumor perfusion increased by 66% RFBV and 58% MRR. While these data clearly demonstrate the inhibitory effects of sunitinib treatment on tumour perfusion they also highlight the rapid reversibility of the anti-vascular effects of the drug. Further, the study demonstrates the utility of DCE-UHS to investigate effects of intermittent sunitinib therapy on vascular parameters.

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 5236.
Abstract 5251: Endosialin: A novel cell surface therapeutic target for early-stage and late-stage neuroblastoma
Authors: Rouleau Cecile , Smale Robert , Kurtzberg Leslie , Weber William , Jones Craig , Roth Stephanie , Bormann Christy , Dunham Sarah , Krumbholz Roy , Curiel Maritza , Wallar Gina , Fu Yao-Shi , Horten Bruce , Gianolio Diego , Schmid Steven , Miller Glenn , Teicher Beverly A.
Published: February 2011

Hypothesis: Endosialin emerged recently as a potential marker and therapeutic target for adult and pediatric sarcoma. Given evidence of a possible common progenitor cell for mesenchymal and neural cell lineages, we wondered whether expression of endosialin may be shared by sarcomas and neuroblastomas, which are cancers of mesenchymal origin and neural crest origin, respectively.

Methods: Endosialin protein expression was studied in live human neuroblastoma cell lines by flow cytometry using a fully human monoclonal antibody against human endosialin. Endosialin-positive human neuroblastoma cells were subsequently implanted at different anatomic sites in nu/nu mice and allowed to grow. Tumors were collected, formalin fixed and subjected to immunohistochemistry using a fully human monoclonal antibody against human endosialin. Using the same IHC assay, endosialin protein expression was also studied in formalin-fixed paraffin-embedded human clinical specimens of neuroblastoma.

Results: We tested 10 human neuroblastoma cell lines for endosialin protein expression by flow cytometry and found that 9/10 expressed endosialin. Several of the positive cell lines were derived from bone marrow metastases, suggesting that endosialin expression is maintained in advanced disease. We modeled endosialin-positive neuroblastoma in vivo by implanting SK-N-AS cells in mice subcutaneously and in the subrenal capsule, kidney being a site where neuroblastoma sometimes originates. Immunohistochemical analysis revealed that SK-N-AS cells, which are positive for endosialin in vitro and derived from the bone marrow metastasis of an adrenal primary tumor, formed endosialin-positive subcutaneous tumors and endosialin-positive renal tumors, demonstrating that endosialin expression is supported by different microenvironments in various anatomic locations.

Immunohistochemistry of human clinical specimens of neuroblastoma showed expression of endosialin. All specimens of neuroblastoma were bone marrow metastases, demonstrating that endosialin is expressed in advanced disseminated neuroblastoma.

Conclusions: Our work demonstrates that endosialin expression is shared by sarcomas and neuroblastomas. The expression of endosialin in neuroblastoma potentially opens up a new therapeutic horizon for neuroblastoma patients, including those suffering from advanced disease.

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 5251.
Abstract 5257: Preclinical evaluation of lestaurtinib (CEP-701) with the retinoids for neuroblastoma
Authors: Norris Robin E. , Minturn Jane E. , Brodeur Garrett M. , Maris John M. , Adamson Peter C.
Published: February 2011

Purpose

Lestaurtinib (CEP-701), a selective and potent inhibitor of the Trk family of receptor tyrosine kinases, has undergone initial clinical evaluation in children with relapsed neuroblastoma. We studied the interaction of CEP-701 with 13-cis retinoic acid (13cRA) and fenretinide (4HPR), two retiniods that have been studied in children with high-risk neuroblastoma.

Methods

In a panel of 5 neuroblastoma cell lines (CHP-134, IMR-5, E6-NBLS, SH-SY5Y, and the SH-SY5Y-BR6 line that has been engineered to express high levels of functional TrkB receptors), in vitro cytotoxicity was assessed following a 72-hour drug exposure using the sulforhodamine B assay. When appropriate, the combination index (CI) of Chou-Talalay was used to characterize the interaction of 13cRA (non-constant ratio) or 4HPR (constant ratio) with CEP-701.

Results

The median (range) IC50 of single agent CEP-701 across all cell lines was 0.09 (0.08-0.17) μM. The combination of 13cRA and CEP-701 resulted in additive to synergistic interactions in 4 of the 5 cell lines studied. Addition of 1 and 5 µM of 13cRA decreased the median (range) CEP-701 IC50 1.5-fold (1.1 to 2.8-fold) and 1.7-fold (1.5 to 1.8-fold), respectively. With 10 µM 13cRA, less than 50% of cells survived when combined with various concentrations of CEP-701. The combination of 4HPR and CEP-701 trended toward being antagonistic, with a median (range) CI at the ED50 of 1.3 (1.1-1.5).

Conclusions

The combination of 13cRA and CEP-701 was additive or synergistic in a spectrum of neuroblastoma cell lines, suggesting that these agents can be used together to eliminate minimal residual disease following intensive chemoradiotherapy for high-risk 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 5257.
Abstract 5261: Transfection of nerve growth factor cDNA into SK-N-BE(2)C neuroblastoma modulates TrkA and p75 receptor expression and activation
Authors: Holub Janette L. , Lautz Timothy B. , Chu Fei , Clark Sandra H. , Madonna Mary Beth
Published: February 2011

The diverse biological role of nerve growth factor (NGF) as a necessary modulator of neuroectodermal cell survival, proliferation and differentiation has been previously well described in both normal and neoplastic cell lines. However, recent studies demonstrate that neuroectodermal tumors, such as neuroblastoma, contain genetic alterations that result in limited secretion of active NGF, as well as expression abnormalities of its receptors TrkA and p75. To better define the differential effects of exogenous and endogenously synthesized NGF, the in vitro response of wild-type and genetically engineered neuroblastoma cells was investigated. Replication-defective NGF-expressing retroviral vectors were prepared using the calcium phosphate co-precipitation method and used to transfect SK-N-BE(2)C cells. Presence of the NGF cDNA was confirmed by RT-PCR, and secretion of NGF into the conditioned media by western immunoblotting. NGF transfection resulted in decreased cell proliferation as determined by nonradioactive cell proliferation assay. Wild type and NGF transfected (BE(2)C-NGF) cells were then incubated with exogenous NGF at 200ng/mL for varying time periods up to 24 hours. The expression of TrkA and p75, as well as proteins of their associated survival and apoptosis cascades were determined by Western immunoblotting of whole cell lysates. p75 expression in the NGF transfected cell line was equivalent to the parental wild type cell, but TrkA expression was significantly lower in BE(2)C-NGF. At baseline, BE(2)C-NGF cells demonstrate significant down-regulation of phospho-cRAF and its downstream effector phospho-p42/44 in comparison to the wild type cell line. Additionally, NGF stimulation further down-regulated phospho-c-RAF expression in the NGF transfected cell line, with maximal down-regulation occurring at 24 hours. BE(2)C-NGF cells also demonstrate significant baseline activation of phospho-p38 in comparison to the wild type cell line, with expression further up-regulated with NGF stimulation at 5 minutes and 1 hour. In conclusion, nerve growth factor cDNA gene transfer into wild type SK-N-BE(2)C neuroblastoma results in decreased activation of the p-42/44 survival pathway and concomitant up-regulation of the p-38 apoptotic pathway, leading to a overall decreased rate of proliferation. This effect is likely the result of a decreased TrkA to p75 receptor ratio, and further understanding of these pathways may facilitate the discovery of novel therapeutic targets for neuroblastoma 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 5261.
Abstract 5268: Stimulated metastasis after treatment with epigenetic drugs through up-regulation of Ezrin
Authors: Yu Yanlin
Published: February 2011

Stimulated metastasis after treatment with epigenetic drugs through up-regulation of Ezrin

Yanlin Yu, Pingyao Zeng, Jingbo Xiang, Shelley L. Berger and Glenn Merlino

Laboratory of Cancer Biology and Genetics, National Cancer Institute, National Institutes of Health, Bethesda, Maryland

The metastases at distant sites are still the main cause of death in cancer patients. New prognostic markers are urgently needed to assess the risk for developing metastasis, and novel therapeutic strategies are needed to treat this deadly disease. Epigenetic drugs such as histone deacetylase inhibitors (HDACis) and inhibitors of DNA methylation are emerging as a new class of anticancer agents and are in several ongoing patient treatment regimens. However, those drugs are thought to be “dirty drugs”, affecting many pathways. Previously, we identified Ezrin as a critical regulator of metastasis in a mouse model of the pediatric cancer rhabdomyosarcoma (RMS) (Yu, et al. Nature Medicine, 2004; Yu, et al. Cacner Res, 2006). In the current study, we examined the status of histone modification at the Ezrin gene chromatin locus and of methylation at the Ezrin promoter in highly and poorly metastatic RMS cell lines. Our results showed that RMS cells with high-level Ezrin and high metastatic potential have elevated acetylation of histone H3 lysine 9 (H3K9) and less methylation at the Ezrin promoter. In contrast, RMS cells with low-level Ezrin and poor metastatic potential have reduced acetylation of H3K9 and elevated methylation at the Ezrin promoter. Our data demonstrate that epigenetic covalent modifications to histones within nucleosomes of the Ezrin gene promoter are linked to Ezrin expression, and suggest that epigenetic regulation may affect the expression of Ezrin, thereby regulating metastasis. We further found that treatment with HDACi TSA or DNA demethylating agent 5-aza can restore expression of Ezrin and stimulate the metastatic potential of poorly metastatic cells with low Ezrin levels. Notably, the combination of Ezrin shRNA and HDACi can significantly inhibit this metastasis. This finding provides new mechanistic and therapeutic insights of epigenetic drugs in cancer 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 5268.
Abstract 527: Pyrrole-Imidazole polyamide inhibits MMP-9 transcription and induces suppression of tumor metastasis
Authors: Wang Xiaofei , Nagase Hiroki
Published: February 2011

Pyrrole-Imidazole (PI) polyamide that targets the AP-1-binding site of the MMP-9 promoter was designed and synthesized as a gene-silencing agent for tumor metastases. MMP-9, the 92-kDa type IV collagenase, contributes to tumor invasion and metastases, and strategies to down-regulate its expression could ultimately be of clinical utility. The synthesized MMP-9 PI polyamide showed selective DNA binding ability and significantly inhibited MMP-9 mRNA and protein expression and enzymatic activity in both MDA-MB-231 and Hela cells. Furthermore, the MMP-9 PI polyamide inhibited migration and invasion by in vitro wound-healing and matrigel-invasion assay. The FITC-labeled PI polyamide was localized in nuclei in 45 minutes of incubation with an MDA-MB-231 cell and remained in the nuclei up to 96 hours after incubation in vitro. It was also quickly localized in the mouse cellular nuclei of many tissues, including liver, kidney and spleen, after intravenous injection without using any drug-delivery system. Moreover, the polyamide treatment significantly decreased metastasis of xenografted tumor cells and host angiogenesis in tumors in a mouse model of liver metastasis. Our results suggest that this PI polyamide that targets the MMP-9 gene promoter can be a novel MMP-9 down-regulating molecule for anti-metastasis.

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 527.
Abstract 5270: Activation of B-raf by TCDD effects on chemotherapeutic resistance and potent inhibition of TCDD mediated tumor invasion of papillary thyroid cancer cells by PD98059
Authors: Ahn Kwang-Sung , Chae Yang Seok , Won Nam Hee , Kim Hyun Gi
Published: February 2011

The BRAF mutation (V599E) is the most common oncogenic genetic alteration in PTC and, through aberrant activation of the MAPK pathway, can initiate the development and promote the progression of PTC. Constitutive activation of the MAPK pathway through genetic alterations, including RAS and B-type RAF (BRAF) mutations, is common in human cancers and is associated with cell malignant transformation and aggressiveness, implicating that targeted inhibition of the MAPK pathway may potentially be an effective therapy for human cancers. AhR receptor ligands such as TCDD are known to activate mitogen activated protein (MAP) kinases and stress-activated protein kinase/c-Jun NH2-terminal kinase. Here we report that PD98059 successfully inhibits cisplatin-induced apoptosis. And induction of phospho-JNK and phospho-ERK-1/2 were noted when SNU-790 cells were treated with 1 nM TCDD. In in vitro tumor invasion assay, TCDD-induced tumor invasion was reduced by either PD98059 or JNK II inhibitor. PD98059 effectively inhibited TCDD-induced tumor invasion when compared to control. The transcription luciferase reporter system demonstrated that PD98059 effectively reduced AP-1 activity when compared to control. The levels of MMP-2 and MMP-9 mRNA expression were reduced slightly by treatment with PD98059. We conclude a significant association of tumor progression in BRAF mutant PTC with the exposure of TCDD. Our findings suggest that MEK inhibitor (PD98059) may function as a small molecule inhibitor of tumor invasion and may provide novel mechanistic insights into the potential therapy for human ATC.

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 5270.
Abstract 5275: Alternate mechanisms of PI3K pathway activation among subtypes of ductal carcinoma in situ suggest distinct pathways for progression to invasive disease
Authors: Sakr Rita , Chandarlapaty Sarat , Barbashina Violetta , Andrade Victor , Heguy Adriana , Morrogh Mary , Olvera Narciso , Paik Wooyul , Arroyo Crispinita , Morrow Monica , Rosen Neal , King Tari
Published: February 2011

INTRODUCTION: Activation of the phosphatidylinositol 3-kinase (PI3K)-AKT pathway via kinase mutations or loss of PTEN is frequently reported in invasive breast cancer and laboratory models suggest that these “second hits” promote invasiveness in HER2 amplified disease. The role of PI3K pathway activation in the transition from in situ to invasive disease remains uncertain. The aim of this study was to assess the frequency of activating lesions in the PI3K pathway in pure high grade DCIS and DCIS associated with HER2 amplified invasive cancer.

METHODS: 89 cases of pure high grade DCIS and 73 cases of DCIS associated with HER2 amplified (HER2+) invasive cancer were identified. Sections from FFPE blocks were used for IHC, FISH and DNA extraction. Pure DCIS, DCIS adjacent to invasive and matched invasive cancers were microdissected for multiplex array (Sequenom®) genotyping of PIK3CA and AKT. IHC was performed for HER2 and PTEN. HER2+ was confirmed by FISH. PTEN was scored relative to adjacent normal cells (scale 0-2).

RESULTS: Among 89 cases of pure high grade DCIS, 51 (57%) were HER2+. PIK3CA mutations were identified in 13/73 (18%) HER2+ invasive lesions and in 11/13 cases the mutation was also present in the adjacent DCIS (11/73 (15%)). PIK3CA mutations were less common in pure HER2+ DCIS (2/51 (4%)) and pure HER2 neg DCIS (2/38 (5%)). Mutations in AKT1 were not identified HER2+ invasive lesions or the adjacent DCIS, yet were found in 3 cases of pure DCIS (2 HER2 neg, 1 HER2 +). PTEN loss was rare in HER2+ invasive lesions (4%), adjacent HER2+ DCIS (5%) and pure HER2+ DCIS (4%) but was seen in 7/38 (18%) cases of pure HER2 neg DCIS. Concomitant loss of PTEN and PIK3CA mutation was seen in 1 case of pure HER2 neg DCIS.

CONCLUSIONS: PIK3CA mutations were observed with similar frequency in HER2+ invasive cancers and in the adjacent DCIS and were uncommon in pure DCIS, suggesting that mutational activation of PI3K may cooperate with HER2 in disease progression. PTEN loss was more common in HER2 neg DCIS and may not be mutually exclusive with PIK3CA mutations, suggesting that these mechanisms may interact in the progression of HER2 neg disease. AKT1 mutations were rare in pure DCIS and not identified in matched HER2+ DCIS and invasive cancer. These data support a role for activation of the PI3K pathway in the transition from in situ to invasive disease and suggest that the mechanism of activation may vary by HER2 status. Further work in HER2 neg DCIS and matched HER2 neg invasive cancer is underway.

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 5275.
Abstract 5283: A role for mitochondrial derived reactive oxygen species in regulating pro-migratory signaling pathways via p130cas
Authors: Hempel Nadine , Melendez J. Andres
Published: February 2011

A cell's migratory and invasive potential depends on a multitude of signaling pathways, which present novel targets for anti-cancer therapies. We and others have shown that reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), are modulators of tumorigenicity and contribute to the migratory phenotype of cancer cells (Connor et al. 2008, Cancer Research). Intrinsic levels of ROS are elevated in numerous metastatic cancer types, which suggests that these cells have evolved to utilize sublethal increases in ROS levels as novel second messengers in regulating pro-metastatic signaling events. Here we present evidence to support this hypothesis and describe how ROS may act as second messengers in the Focal Adhesion Kinase (FAK) pathway. Cells displaying intrinsic picomolar changes in H2O2 levels, either endogenously (metastatic 253J-BV bladder cancer cell line, compared to its non-metastatic parental 253J-P line), or as a consequence of enforced mitochondrial manganese superoxide dismutase (Sod2) expression, display an enhanced migratory phenotype. This is accompanied by a loss of mature vinculin positive focal adhesions. Mechanistically, we show that the protein tyrosine phosphatases PTP-PEST and PTEN are oxidized and inactivated in cells displaying enhanced H2O2 levels and that this can be reversed by effective removal of H2O2 following expression of the H2O2 detoxifying enzyme catalase (CAT). A consequence of PTP-PEST oxidation and inactivation is a concomitant increase in phosphorylation of its primary substrate p130cas, an adaptor protein of the FAK signaling cascade. Further, increases in intracellular H2O2 levels shift the localization of p130cas to the cell surface, as assessed by membrane/cytoskeletal fractionation and immunofluorescent staining, indicative of localized activation of FAK pathway signaling at the leading edge. Again co-expression of CAT negates these effects, indicating that these mechanisms are H2O2 dependent. The data described support the hypothesis that elevated mitochondrial ROS levels can regulate the spatial activation of pro-migratory signaling pathways, enhancing the metastatic potential of cancer 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 5283.
Abstract 5294: IIp45 inhibits cell migration through inhibition of HDAC6
Authors: Zhang Wei , Song Sonya W. , Sun Jiyuan , Bruner Janet , Fuller Gregory
Published: February 2011

IIp45 (aka MIIP) is a newly discovered gene whose protein product inhibits cell migration. HDAC6 is a class IIb deacetylase that specifically deacetylates α-tubulin, modulates microtubule dynamics and promotes cell migration. A yeast two-hybrid assay using IIp45 as the bait identified HDAC6 protein as a binding partner of IIp45. This physical interaction of the two functionally antagonizing proteins was confirmed by Glutathione S-transferase (GST) pull-down assay and co-immunoprecipitation (Co-IP) assay in human cells. Serial deletion constructs of HDAC6 were used to characterize the interaction of HDAC6 and IIp45, and this analysis found that the two catalytic domains of HDAC6 protein were required for IIp45 binding. We examined the protein expression patterns of IIp45 and HDAC6 in glioma tissues. Elevated protein levels of HDAC6 were found in high grade glioma samples in contrast to the decreased protein expression of IIp45. The potential negative regulation of HDAC6 expression by IIp45 was confirmed in cell lines with altered IIp45 expression by constitutive overexpression or small interfering RNA knockdown. Protein turnover study revealed that overexprsssion of IIp45 significantly reduced intracellular protein stability of endogenous HDAC6, indicating a possible mechanism for the negative regulation of HDAC6 by IIp45. Results from the HDAC activity assay demonstrated that overexpressed IIp45 effectively decreased HDAC6 activity, increased acetylated α-tubulin, and reduced cell migration. The increased cell migration by siIIp45 knockdown was significantly reversed by co-transfection of siHDAC6. Thus, we report here for the first time a novel mechanism by which IIp45 inhibits cell motility through inhibition of HDAC6

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 5294.
Abstract 5295: S100A4 regulates tumor growth, motility, and invasion in pancreatic cancer
Authors: Sekine Hitoshi , Tabata Takahiro , Tsukamoto Nobukazu , Yamamura Akihiro , Abe Keiko , Yoshino Yuki , Sato Keisuke , Chen Na , Umetsu Yukiko , Sato Daisuke , Gu Zhaodi , Fukushige Shinichi , Shima Kentaro , Motoi Fuyuhiko , Egawa Shinichi , Sunamura Makoto , Horii Akira
Published: February 2011

S100A4 protein belongs to the S100 subfamily, which has grown to be one of the large subfamilies of the EF-hand Ca2+-binding proteins, and overexpression of S100A4 is suggested to associate with cell proliferation, invasion, and metastasis. We observed frequent overexpression of S100A4 in pancreatic cancer cell lines by quantitative real-time RT-PCR and Western blotting. Tissue microarray analysis validated S100A4 overexpression in primary pancreatic cancer tissues, and overexpression of S100A4 associated with perineural invasion. We further analyzed RNAi-mediated knockdown to address the possibility of its use as a therapeutic target for pancreatic cancer. The specific knockdown of S100A4 strongly suppressed cell growth, induced G2 arrest and eventual apoptosis, and decreased cell migration. Furthermore, microarray analyses revealed that knockdown of S100A4 induced expression of the tumor suppressor genes PRDM2 and VASH1. We further established stably S100A4-expressing cell lines using two pancreatic cancer cell lines with low level of S100A4 expression; remarkable accelerated cell growth and motility was observed after induction of S100A4, whereas no change was observed in the control cells. Comparison of the expressional profiles after siRNA-mediated knockdown and expression vector-mediated induction is underway, and the results will also be demonstrated and discussed.

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 5295.