bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2310: Down-regulating BCL-2 gene expression in a resistant GIST cell line by a RNA G-quadruplex interacting small molecule ligand
Authors: Parkinson Gary N. , Gunaratnam Mekala , Li Xiong Wei , Collie Gavin , Reszka Anthony P. , Todd Alan R. , Fletcher Jonathan A. , Neidle Stephen
Published: June 2012

We have previously shown that KIT protein expression in the gastrointestinal tumour (GIST) cell line GIST882 can be down-regulated using a small molecule approach that targets expression at the transcriptional level. The small-molecule naphthalene diimide derivative ND1 developed as a G-quadruplex telomere targeting agent can inhibit telomerase activity and can also reduce c-kit mRNA levels, consistent with a dual G-quadruplex mechanism of action in this cell line. An Imatinib (Gleevec) resistant patient-derived cell line GIST48 expresses high levels of the anti-apoptotic protein bcl-2. The compound ND1 down-regulates the expression of bcl-2 in this resistant cell line, while having no discernable effect on c-kit protein expression levels or telomerase activity, both associated with DNA quadruplex stabilization. Instead we observe that translation of the apoptosis-related protein is impeded, while mRNA levels remain unaffected. The identification of a G-rich quadruplex-forming sequence in the 5’-UTR region of the bcl-2 gene, upstream of the translation start site, lead us to the development of a dual luciferase reporter assay. Utilizing a psiCHECK-2 vector we inserted the G-rich 67 base pair bcl-2 5’-UTR gene sequence upstream of the Renilla luciferase start codon. In vitro experiments have shown that the G-rich sequence can form a stable RNA G-quadruplex and interfere with translation. Our in vivo transfection experiments with ND1 in a GIST cell line shows concentration-dependent inhibition of bcl-2 protein expression. RNA G-quadruplex formation and ligand stabilization may thus be a novel anti-cancer route for the down-regulation of the anti-apoptotic protein bcl-2 in chemo-resistant cancers.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2310. doi:10.1158/1538-7445.AM2011-2310
Abstract 2321: Pyrrolobenzodiazepine (PBD) antitumour agents as chemical probes to determine the number of base pairs required for DNA minor groove structure
Authors: Rahman Khondaker M. , Hijon Isabel G. , James Colin H. , Thurston David E.
Published: June 2012

The pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) are sequence-selective covalent-binding DNA-interactive agents that fit perfectly in the minor groove of double-stranded DNA due to their isohelical 3-dimensional shape. They possess an electrophilic N10-C11 imine (or the equivalent) that can form a covalent aminal bond between their C11-position and the C2-NH2 group of a guanine. One synthetic PBD dimer, SJG-136, has just entered Phase II clinical trials. Previous studies using HPLC/MS, CD spectroscopy, high-field NMR, X-ray crystallography and gel-based assays such as DNA footprinting and in vitro transcription stop are all consistent with the requirement for duplex DNA for covalent binding. Conversely, it is well-documented that PBDs will not interact with single stranded DNA which lacks minor groove structure. In this study, these observations served as a basis to use PBDs as chemical probes to determine the minimum number of bases required to create a minor groove environment within a DNA duplex. Thus we studied reaction of the naturally-occurring anthramycin (3 base pair span) and the synthetic C8-linked PBD bis-pyrrole conjugate GWL-78 (6 base pair span) with a range of designed duplex oligonucleotides of increasing length (3 to 11 base pairs), each containing an AGA binding triplet. Reaction was monitored by HPLC/MS which provided data on both the rate and extent of reaction, and the structure of DNA adducts formed. For both anthramycin and GWL-78, we found that a minimum of 7 DNA base pairs were required for reaction to occur, suggesting that this may represent the minimum necessary to provide a suitable minor groove environment. Furthermore, we found that the rate of reaction for both PBDs increased with the length of the oligonucleotide between 7 to 10 base pairs. It is noteworthy that GWL-78 is approximately twice the length of anthramycin but both require at least 7 base pairs in order to covalently bind. This suggests that both may be responding to the formation of a minor groove environment in an oligonucleotide that reaches 7 base pairs in length, rather than a direct relationship between the length of the PBD and the number of base pairs in the oligonucleotide. These observations were supported by molecular modeling which confirmed that 7 base pairs are required to provide minor groove structure. This is the first study to use a chemical probe of this type to monitor formation of a minor groove. This information should be useful in the design of future generations of PBDs and similar DNA-interacting anticancer agents.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2321. doi:10.1158/1538-7445.AM2011-2321
Abstract 2326: The first crystal structure of a telomeric quadruplex DNA with a metal-containing ligand
Authors: Campbell Nancy H. , Abd Karim Nurul H. , Parkinson Gary N. , Vilar Ramon , Neidle Stephen
Published: June 2012

The inhibition of telomerase function in cancer cells via induction of a higher-order four-stranded quadruplex arrangement in the telomeric DNA substrate, requires the participation of a quadruplex-stabilizing ligand. We showed several years ago that certain metal complexes could fulfil this role and not only have selectivity for a quadruplex arrangement but had significant biological activity.

We now report on the crystal structures of square-planar nickel (II) and copper (II) complexes containing salphen ligands with a human telomeric quadruplex. The structures show that the metal complexes are bound on an external G-quartet face of the quadruplex, and that the metal itself is positioned above the potassium-containing ion channel of the quadruplex, providing important stabilization.

The planar groups of the ligand are stacked onto guanine bases, and the charged amino side-chains are positioned in the grooves of the quadruplex. These features are enabling the available structure-activity data to be rationalized and will be discussed in terms of the design of future complexes with enhanced activity.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2326. doi:10.1158/1538-7445.AM2011-2326
Abstract 2349: Integrin α6β4 contributes to HGF-stimulated migration and invasion by promoting autocrine EGFR signaling in pancreatic cancer
Authors: Chen Min , Cruz-Monserrate Zobeida , Knifley Teresa , Davis Kelley A. , O'Connor Kathleen L.
Published: June 2012

The lethality of pancreatic cancer is due to elevated incidences of invasion and metastasis. Previous studies from our lab and others show that pro-invasive integrin α6β4 is upregulated in early stage pancreatic cancer, maintained during tumor progression and, in turn, promotes chemotactic migration and invasion toward HGF. To gain insight into the mechanisms governing how integrin α6β4 promotes these processes, we performed Affymetrix Gene Chip analysis on Panc-1 clones with differing levels of integrin α6β4 expression. After statistical processing, we found 582 genes altered by integrin α6β4 signaling. From these results, we found that high integrin α6β4-expressing Panc1 clones substantially enhanced expression of genes affecting epidermal growth factor receptor (EGFR) signaling. The genes upregulated by integrin α6β4 included EGFR, EGF-like ligands amphiregulin (AREG) and epiregulin (EREG), ectodomain cleavage proteases ADAMTS1, MMP1, and hyaluronan synthase 3. The upregulation of these genes was confirmed by real-time PCR, immunoblot analysis and/or ELISA. Stable overexpression of integrin β4 in low integrin β4 expressing cells increased the levels of AREG and EREG by real-time PCR. We further tested the role of autocrine EGFR signaling by pretreating cells with EGFR blocking antibodies (LA1) or EGFR-specific protein tyrosine kinase inhibitor (PD153035) and tested HGF-stimulated chemotaxis, chemo-invasion and invasive growth in three-dimensional culture. Interestingly, these inhibitors blocked integrin α6β4-dependent HGF-stimulated chemotaxis, invasion and invasive growth. In summary, our data suggest integrin α6β4 confers a motile and invasive phenotype to pancreatic carcinoma cells, in part, by coordinately regulating expression of key elements of autocrine EGFR signaling.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2349. doi:10.1158/1538-7445.AM2011-2349
Abstract 2352: GSK3b can phosphorylate Slug and modulate cancer metastasis
Authors: Kao Shih-Han , Wang Wen-Long , Hong Tse-Ming , Yang Pan-Chyr
Published: June 2012

Metastasis is a process complicated by multiple factors and genetic events. Slug, a Snail/Slug family transcription factor, promotes epithelial-mesenchymal transition (EMT), a process pivotal in development and tumor invasion and migration. Thus, how to regulate Slug may be useful in treating or preventing tumor metastasis in cancer. Glycogen synthase kinase-3β (GSK-3β) is a Serine/Threonine selective kinase and may regulate a broad range of cellular functions. It has been shown that GSK-3β is mostly inactivated in epithelial cancers and hence may enhance the stability of various oncogenic transcription factors. Recently, we have found that GSK-3β can interact with Slug protein, implying Slug as a possible phosphorylation substrate of GSK-3β. We confirm that GSK-3β can phosphorylate Slug by mutating the consensus motif of GSK-3β phosphorylation sites within the Slug protein (S/TxxxS/T) in the in vitro kinase assay. The cycloheximide-chase assay showed that the phosphorylation mutant (4SA) was more stable than the wild-type counterpart (wtSlug). Moreover, the wtSlug protein became more stable in the presence of kinase-dead GSK-3β or when GSK-3β was knocked down, suggesting that Slug may be more labile under GSK-3β phosphorylation. To further study whether the functions of Slug are affected by GSK-3β-induced phosphorylation, we carried out the luciferase assay and the preliminary data show that the 4SA mutant retains the transcription repression activity but is less sensitive to GSK-3β treatment. The downstream effector of EMT, E-cadherin, was repressed in HEK293/wtSlug and HEK293/4SA. More functional assays are needed to confirm that GSK-3β-induced Slug phosphorylation is involved in cancer progression. Overall, this study may provide the molecular mechanism by which the GSK-3β/Slug pathway regulates cancer cell migration, invasion and metastasis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2352. doi:10.1158/1538-7445.AM2011-2352
Abstract 2353: Fibulin-3, an extracellular matrix protein, regulates Notch signaling and promotes brain tumor cell invasion and survival
Authors: Hu Bin , Agudelo Paula A. , Saldivar Joshua C. , Sim Hosung , Dolan Claire E. , Mora Maria E. , Nuovo Gerard J. , Cole Susan E. , Viapiano Mariano S.
Published: June 2012

Malignant gliomas are the most common primary brain tumors and have extremely poor prognosis. Glioma cell infiltrate into normal neural tissue, escaping conventional chemo-radiotherapy and making tumor relapse almost unavoidable. We have recently characterized a novel extracellular matrix (ECM) protein named fibulin-3, which is highly expressed in gliomas but is absent in normal brain and rarely expressed in other solid tumors. We demonstrated that fibulin-3 regulates the expression of metalloproteases and promotes glioma cell invasion. In addition, this ECM protein increases the resistance of glioma cells to pro-apoptotic treatments, including starvation, oxidative stress and treatment with temozolomide. To further understand the mechanisms of fibulin-3 in gliomas, we investigated several candidate pathways but found no activation of EGFR, MAPK or Akt signaling. Interestingly, fibulin-3 bears significant homology with the Notch ligands from the Delta-like family, which prompted us to study the possible activation of this pathway, which is a critical anti-apoptotic pathway in cancer cells. Surprisingly, we found that fibulin-3 increased cleavage of endogenous Notch-1 in glioma cells as well as activation of a Notch-dependent reporter. In agreement, fibulin-3 expression regulated the levels of the Notch effector Hes-1 both in vitro and in vivo. Moreover, the pro-invasive effect of fibulin-3 in glioma cells was inhibited by pharmacological inhibition of Notch signaling or knockdown of Notch-1. This is potentially a novel mechanism by which tumor-associated ECM molecules can activate Notch signaling and modulate this key pro-tumoral pathway in gliomas. These results underscore the potential of fibulin-3 as a glioma-specific target in the tumor microenvironment.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2353. doi:10.1158/1538-7445.AM2011-2353
Abstract 2365: Actopaxin: A novel regulator of cell migration and invasion in human hepatocellular carcinoma
Authors: Ng Lui , Poon Ronnie , Wong BCY , Pang Roberta
Published: June 2012

Background: The mortality rate of hepatocellular carcinoma (HCC) is high due to tumor recurrence and development of metastasis, involving cell spreading, lamellipodia formation and cell migration. Formation of focal adhesions is a key regulator of cell motility and cell invasion. Actopaxin, a focal adhesion and cytoskeleton-associated protein, is required for such processes. The aim of this study was to examine the role of Actopaxin in HCC cell migration, invasion and development of metastasis.

Methods: The expression of Actopaxin in primary and metastatic liver cancer cell-lines was examined by western blot and RT-PCR. The functional and molecular effects of Actopaxin expression were studied by transient as well as stable enforced expression or down-regulation in HCC cell-lines. Immunofluorescence staining for actin cytoskeleton and focal adhesion was performed to examine the effect of Actopaxin expression on cell shape, stress fiber organization, and focal adhesion. The clinicopathological significance of Actopaxin expression in tumorous tissues of 119 HCC patients was also investigated.

Results: High protein level of Actopaxin was found in the metastatic HCC cell-lines compared with other non-metastatic HCC cell-lines. Expression of a shorter form of Actopaxin was also detected in some of these cell-lines. Comparing to Actopaxin (LF-Actopaxin), the unreported short form (SF-Actopaxin) lacks a fragment in the C-terminal and hence results in an incomplete 2nd CH domain which consists binding sites for its downstream activation targets. Expression of LF-Actopaxin but not SF-Actopaxin in HCC cell-lines was positively correlated with their migration and invasion potential in vitro, accompanied by altered expressions of several focal adhesion proteins as well as EMT proteins. Study in clinical samples showed that LF-Actopaxin expression was positively correlated with tumor size (p<0.05) while SF-Actopaxin expression was lower in HCC tumor compared with corresponding nontumorous liver (p<0.001) and lower in HCC with metastasis compared with HCC without metastasis (p<0.05).

Conclusion: This study demonstrated for the first time the pro-migratory effects of Actopaxin in human HCC, and the existence of SF-Actopaxin which displays different expression pattern as well as functional and molecular effects.

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Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2365. doi:10.1158/1538-7445.AM2011-2365
Abstract 2366: Impact of EWS-FLI induced changes in gene expression and cell behavior on metastatic potential
Authors: Chaturvedi Aashi P. , Welm Alana , Lessnick Stephen , Beckerle Mary
Published: June 2012

Ewing's sarcoma (ES) is frequently metastatic at presentation, a condition that is associated with poor prognosis. ES is caused by the oncogenic fusion of the EWSR1 and FLI1 genes to encode a chimeric EWS/FLI protein product that results in an altered transcriptional program. Because the metastatic potential of ES appears to develop at an early stage, we have explored whether the oncogene-driven misregulation of gene expression might contribute directly to promotion of metastasis. Metastasis is a complex process involving sequential and interrelated steps. In this study, we have examined the impact of EWS/FLI expression on cell migration, cell adhesion, and cytoarchitecture of ES cells by comparing the behavior of A673 ES cells with that of A673 cells in which we knocked down expression of EWS/FLI by RNA-interference. In contrast with what one might expect for a critical oncogene, we found that EWS/FLI expression reduced in vitro migration and Matrigel invasion. Consistent with these findings, expression of EWS/FLI abrogated cell adhesion and spreading. An in vivo mouse lung colonization assay revealed that ES cells treated with EWS/FLI RNAi adhere and colonize better in the lung tissue compared to ES cells, further supporting the in vitro adhesion results. At the cellular level, adhesion strength is correlated with the capacity of cells to form focal contacts and stress fibers. Knockdown of EWS/FLI expression in ES cells resulted in a striking remodeling of the actin cytoskeleton, with establishment of robust stress fibers and focal adhesions. Gene expression profiles, Western immunoblot analysis, and immunofluorescence staining confirmed that EWS/FLI expression is associated with reduced levels of focal adhesion proteins, including zyxin, a key regulator of actin assembly. Introduction of a zyxin cDNA expression construct into A673 ES cells was sufficient to restore the integrity of the actin cytoskeleton and focal adhesions, and to partially restore cell adhesion, both in vitro and in vivo, demonstrating a central role for zyxin in regulation of ES phenotype. Since release of cells from the primary tumor site is a critical initial step in metastasis, the profound impact of EWS/FLI expression on cytoarchitecture and cell adhesion may contribute to the metastatic phenotype in a unique, yet fundamental, way. Based on these data, we hypothesize that EWS/FLI-induced changes in gene expression simultaneously promotes both oncogenic transformation and tumor cell dissemination.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2366. doi:10.1158/1538-7445.AM2011-2366
Abstract 2371: Comparison of repeated minor liver resection and major resection on lung metastasis growth in mice
Authors: Momiyama Masashi , Suetsugu Atsushi , Tome Yasunori , Bouvet Michael , Chishima Takashi , Endo Itaru , Hoffman Robert M.
Published: June 2012

Liver resection has traditionally been the first choice treatment for patients presenting with primary and metastatic liver tumor. Many of the factors up-regulated during liver regeneration after a liver resection have been implicated in tumor growth and the extent of the up-regulation of these factors correlate with the degree of liver resection. However, the effects of a major liver resection compared to repeated minor resections on the growth of malignant tumor are unknown. This study examines the effects of liver regeneration on the growth of the lung metastatic tumors in an established mouse model, after major (70%) and repeated minor (35%+35%) liver resection. Experimental lung metastases were established by tail vein injection with the Colon 26 murine adenocarcinoma cell line expressing green fluorescent protein (GFP). Three days after cancer cell injection, groups of mice underwent liver resection (35%+35% vs. 70%). Lung tumor growth was measured by fluorescence imaging with the Olympus OV100 Small Animal Imaging System. Although, major and repeated minor resection removed the same volume of liver parenchyma, the two procedures had very different effects on lung metastases growth: Major resection stimulated lung metastatic growth. In contrast to major resection, repeated minor resection significantly inhibited the growth of metastatic lung tumors. There was no significant difference in liver regeneration between the two groups. These results show that repeated minor resection of liver is much safer regarding tumor stimulation than major resection. The results of this study indicate that when liver resection is necessary, repeated minor liver resection is superior to major liver resection, since repeated minor resection inhibits lung metastasis and major resection stimulates lung metastasis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2371. doi:10.1158/1538-7445.AM2011-2371
Abstract 2374: First time identification of circulating tumor cellsin murine models of human non-small cell lung cancer
Authors: Mordant Pierre , Loriot Yohann , Decraene Charles , Lahon Benoit , Castier Yves , Soria Jean-Charles , Deutsch Eric
Published: June 2012

Background. Circulating tumor cells (CTCs) have been found in up to 40% of patients with either localized or metastatic non-small cell lung cancer (NSCLC). However, level of CTCs has never been established in preclinical murine models of human NSCLC. We sought to determine the levels of CTCs in subcutaneous and orthotopic murine models of human non-small cell lung cancer.

Material and Methods. We used athymic nude mice and luciferase-positive A549 lung adenocarcinoma cell line (A549-luc, Xenogen Corp, USA). In group 1, animals underwent subcutaneous injection of cells in both flank (n=6). In group 2, animals underwent general anesthesia, chest wall incision, and transpleural injection of cells in the parenchyma of the left lung (n=15). After 2 weeks, bioluminescent in vivo imaging (IVIS) was performed after intra peritoneal injection of luciferine, and xenograft implantation rates were calculated. During the third week, tumor-bearing animals were anesthetized, 1-mL venous blood was punctured from the cavernous sinus, mixed with 7-mL of healthy human blood, and tested using the Cellsearch assay (Veridex LLC, USA). Animals were then humanely killed.

Results. Xenograft implantation rates were 100% in group 1 (n=6) and 60% (n=9) in group 2. Among the 6 blood tubes collected from the first group, none were clotted, 6 tubes underwent successful Cellsearch assay, and 5 assays were positive for CTCs detection (CTCs level range 2-5, mean 2.5, sd 1.76). Among the 9 blood tubes collected from the second group, 3 tubes were clotted and impossible to analyze, 6 tubes underwent successful Cellsearch assay, and 5 assays were positive for CTCs detection (CTCs level range 2-21, mean 9.5, sd 9.35). When comparing groups, the difference in CTC levels show a trend toward statistical significance (student test, p=0.058).

Conclusions. Two weeks after tumor implantation, CTCs are detected in 83% of nude mice bearing either subcutaneous or orthotopic NSCLC tumors. Evolution of CTCs level should be established, but this model could be of interest to study oncogenesis and to assess new treatments.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2374. doi:10.1158/1538-7445.AM2011-2374
Abstract 238: Inhibition of SHP2 abolishes mammary tumorigenesis in mice
Authors: Matalka Fatimah , Hartman Zachary , Agazie Yehenew M.
Published: June 2012

The Src homology phosphotyrosyl phosphatase 2 (SHP2) plays a positive role in receptor tyrosine kinase-induced mitogenic and cell survival signaling. In addition, SHP2 mediates cell transformation induced by oncogenic tyrosine kinases. Here, we provide evidence that show that the SHP2 protein is overexpressed in basal-type/triple-negative breast cancer (BTBC) with strong correlation to higher tumor grade, metastasis and elevated expression of the epidermal growth factor. We further show that SHP2 plays an essential role in the tumorigenesis and metastasis of BTBC cells (MDA-MB-231 and MDA-MB-468) transplanted into the mammary glands of NOD/SCID mice. These results suggest that SHP2 can be used for targeted therapy in BTBC.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 238. doi:10.1158/1538-7445.AM2011-238
Abstract 2381: Preclinical molecular targeting therapy of pancreatic cancer using a genetically engineered mouse model
Authors: Miyabayashi Koji , Ijichi Hideaki , Mohri Dai , Tada Motohisa , Asaoka Yoshinari , Ikenoue Tsuneo , Omata Masao , Moses Harold L. , Koike Kazuhiko
Published: June 2012

Patients with pancreatic ductal adenocarcinoma have a poor prognosis. New therapeutic approaches to improve the prognosis are expected. We generated pancreas-specific Tgfbr2 knockout mice in the context of Kras activation, which recapitulated clinical and histopathological manifestations of human pancreatic cancer. Using this model we investigated the anti-tumor effects of gemcitabine and molecular targeted drugs.

We treated the mice with EGFR inhibitor elrotinib or VEGFR inhibitor axitinib in combination with gemcitabine. Median survival times were 45, 60, 77, and 74 days for control, gemcitabine alone, gemcitabine + axitinib, and gemcitabine + erlotinib, respectively.

In vitro assays, elrotinib inhibited phosphorylation of EGFR and both drugs also inhibited proliferation of human pancreatic cancer cell lines in vitro.This mouse model also recapitulated chemosensitivity of human pancreatic cancer and suggested erlotinib and axitinib in combination with gemcitabine can be superior to gemcitabine alone. This mouse model helps us to investigate the mechanism of the action of drugs and chemoresistance.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2381. doi:10.1158/1538-7445.AM2011-2381
Abstract 2389: Efficacy of ABT-737 against c-Myc-induced mammary adenocarcinomas in transgenic and syngrafted mouse tumor models
Authors: Eskelinen Vilja , Saarikoski Sirkku T. , Nieminen Anni I. , Haapakorva Elias , Marques Elsa , Klefström Juha
Published: June 2012

Traditional preclinical discovery methods and models only poorly predict drug efficacy in clinical trials. It has been reasoned that present cultured tumor cell line and xenotransplantation-based in vivo models are too reductionist, failing to recapitulate tumour heterogeneity, tumor-microenvironment interactions and tumor microevolutionary trajectories. The genetically engineered mouse models (GEMMs) hold promise as next generation preclinical models, but the caveat is that GEMMs are not straightforward suitable for preclinical cohort studies. Here we hypothesized that mammary tumors from tumor-prone WAP-Myc mice retain their molecular characteristics and authentic drug responses when recreated as syngrafts in the mammary fat pads of recipient mouse. This platform would allow rapid and non-laborious pilot cohort studies with GEMM tumors. We are testing the hypothesis by determining tumor markers and apoptotic efficacy of BH3-mimetic ABT-737 against c-Myc-induced mammary adenocarcinomas and pulmonary metastases in both transgenic and syngraft models. In the WAP-Myc mice c-Myc is induced in the luminal mammary epithelial cells during late pregnancy resulting in development of endogeneous mammary adenocarcinomas in nearly 100% of multiparous animals. We observe that the tumors are transplantable as cells suspensions (25 000 cells/gland) with approximately 90% success rate. While the transgenic WAP-Myc tumors develop with an average of 3.5 months latency, the syngrafted tumors develop within 3-4 weeks post-transplantation. However, in contrast to 20% rate of pulmonary metastases in the transgenic WAP-Myc mice, syngrafted animals need to be sacrificed prior to metastasis formation. Our preliminary results suggest that the tumor histopathology does not significantly change and the tumor heterogeneity is preserved in the syngrafted tumors. To investigate the response of tumors to ABT-737, animals bearing either endogenous or transplanted tumors were treated with 100mg/kg of ABT-737 for 21 days. ABT-737 reduced primary tumor growth and lung metastases as well as enhanced caspase-3 activity in the transgenic WAP-Myc tumors. Also the syngrafted tumors respond to ABT-737 and the data comparing effects in transgenic versus syngrafted tumors will be presented. Our results demonstrating feasibility of the syngraft platform may help to alleviate many shortcomings of GEMMs in preclinical studies.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2389. doi:10.1158/1538-7445.AM2011-2389
Abstract 239: Biomarker profiling and combination studies of receptor tyrosine kinase inhibitors of EGFR, ERBB2, IGFR-1, FGFR and PDGFR
Authors: O'Day Christine L. , Ovechkina Yulia , Marcoe Karen , Keyser Robert , Chesnut-Speelman Jessica , Hnilo James
Published: June 2012

Targeting Receptor Tyrosine Kinases (RTKs) for therapeutic efficacy has been a promising approach for modern oncological agents. However, not all cancers respond to these agents and understanding of the feedback and resistance mechanisms is not complete. We have used an In vitro cellular profiling of 240 genetically characterized tumor human cell lines to help understand genotypes that convey sensitivity and resistance to these agents. While most of the sensitive cell lines were unique to a given RTK inhibitor, many of the resistant cell lines had similar genotypes. We have investigated downstream genes for mutations, over-expression and phosphorylation status. Phosphorylation levels pAKT, pERK, and pJNK, and as well as basal levels of p27KIP, EGFR and ERBB2 protein levels were analyzed. Many of the downstream mutations or activated genes confer resistance to the RTK inhibitors such as AKT, PTEN and PI3K mutations.

In addition to downstream activation, we have found that overexpressed RTKs often seem to confer resistance to other RTK inhibitors. For example, EGFR over-expressed cell lines disproportionately populated the group of cell lines resistant to an IGFR-1 receptor agonist. We have investigated cross talk between these various RTKs. Both EGFR and IGFR-1 inhibitors were tested in a combinatorial approach through 60 cell lines for synergy and antagonism. We evaluate synergy calculations through combination index and bliss analysis across 60 cell lines. Other combinations are also in the process of being tested for synergy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 239. doi:10.1158/1538-7445.AM2011-239
Abstract 2399: Simvastatin inhibits advanced prostate cancer in TRAMP mice
Authors: Drenkhahn Sara K. , Jackson Glenn A. , Huber Christal D. , Browning Jim D. , Fritsche Kevin L. , Wiedmeyer Charles E. , Besch-Williford Cynthia L. , Lubahn Dennis B.
Published: June 2012

Controversial literature suggests the potential off-label benefit of statins in the prevention of breast and prostate cancers. As millions of Americans are currently taking statins to lower cholesterol and prevent heart disease, we wanted to test if simvastatin could inhibit prostate carcinogenesis in the TRAMP (TRansgenic Adenocarcinoma of the Mouse Prostate) model. We hypothesized that simvastatin would inhibit the most aggressive form of cancer in the model, poorly differentiated carcinoma (PDC), similar to what has been seen in epidemiological studies. Mice were fed a Western Diet to mimic the high-fat diet common among men in the United States (n=25 per group). Two additional groups were fed the Western diet supplemented with either 0.025% or 0.050% w/w simvastatin. The control mice on the Western diet had an increase of PDC when compared to a low-fat AIN93 casein based diet (48% vs. 32%). While the 0.025% simvastatin Western diet reduced PDC incidence from 48% to 38%, the 0.050% simvastatin Western diet drastically reduced PDC incidence from 48% to 16% when compared to Western controls (p=0.0153 by Chi square analysis). Changes in serum profiles analyzing total cholesterol, LDL, HDL, and triglycerides did not correlate to the reduction in PDC incidence. In conclusion, our results show that simvastatin can reduce the most aggressive stage of prostate cancer in the TRAMP model and supports the observation that simvastatin reduces the risk of developing advanced prostate cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2399. doi:10.1158/1538-7445.AM2011-2399
Abstract 2404: TMPRSS2, a type II transmembrane serine protease, alters the prostate tumor microenvironment and promotes metastasis
Authors: Sun Yu , Lucas Jared , Hernandez Susana , Nelson Peter
Published: June 2012

Introduction: Extracellular and cell surface proteases play indispensible roles in embryonic development and normal tissue homeostasis. They also contribute to tumor pathology involving cellular invasion and metastasis. As the majority of cancer-related deaths are associated with metastasis, understanding contributors to this process is essential for developing improved treatment strategies. TMPRSS2 is a Type II transmembrane serine protease (TTSP) regulated by androgenic hormones and is expressed preferentially in the human prostate. Recent studies have indicated that TMPRSS2 is expressed highly in primary and metastatic prostate cancers, though functional activities are poorly defined.

Methods: To determine the relevance and consequence of TMPRSS2 overexpression in prostate cancer, we generated four transgenic mouse models that overexpress wild-type or mutant variants of human and mouse TMPRSS2 under the control of the prostate-specific probasin promoter (PB-TMPRSS2). We further crossed PB-TMPRSS2 mice with the LADY model of prostate cancer, and evaluated tumor growth and the impact on metastasis.

Results: We confirmed that the PB-TMPRSS2 transgenic mouse lines have significantly elevated levels of TMPRSS2 protease in prostate epithelium. Although enhanced TMPRSS2 expression did not affect mouse fertility, we observed disorganization and disruption of the basement membrane of prostate glands, regardless of whether the overexpressed enzyme is of human or mouse origin. Immunohistochemical analysis identified dramatically altered levels of β4-integrin and collagen IV in the prostate glands of TMPRSS2-overexpressing mice, with the most significant changes occurring in ventral and anterior lobes. Histological changes in cell differentiation, proliferation or apoptosis were not observed. While a strain of LADY transgenic mice expressing SV40T antigen under the control of the probasin promoter (PB-SV40T) develop prostate cancer without metastatic spread, crosses with PB-TMPRSS2 mice produced offspring with high rates of metastasis to liver, lung, lymph node and bone.

Conclusions: Our study pinpoints a mechanistic pathway by which TMPRSS2 contributes to primary prostate cancer progression and promotes metastasis, paralleling the effects of Hepsin overexpression, a structurally similar protease known to promote metastasis. These results provide support for the development of therapeutic approaches to specifically limit TMPRSS2 proteolytic activity in prostate cancer patients.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2404. doi:10.1158/1538-7445.AM2011-2404
Abstract 2409: A precise orthotopic metastatic model of rectal cancer
Authors: Kishimoto Hiroyuki , Momiyama Masashi , Aki Ryoichi , Kimura Hiroaki , Suetsugu Atsushi , Bouvet Michael , Fujiwara Toshiyoshi , Hoffman Robert M.
Published: June 2012

We have developed a novel technique to orthotopically transplant rectal cancer cells. This model was established by intrarectal transplantation of mouse rectal cancer cells stably expressing fluorescent protein, followed by disrupting the epithelial cell layer of the rectal mucosa by soaking with an acetic acid solution. Early stage tumor was detected on the rectal mucosa from as early as 6 days after transplantation, which then became invasive into the submucosal tissue. The tumor incidence (%) on the rectal mucosa and size (mean volume ± SD) were 100% and 1232.4 ± 994.7 mm3 at 4 weeks after transplantation, respectively. Spontaneous lymph node metastasis and lung metastasis were also observed 4 weeks after transplantation in over 90% of mice. This rectal tumor model to mimic of the natural course of rectal cancer accurately. The model will be more useful for evaluation of the tumor environment, metastasis and agents which suppress or enhance the growth of tumor at early stages as well as metastasis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2409. doi:10.1158/1538-7445.AM2011-2409
Abstract 2418: Establishment and characterization of a primary chordoma xenograft
Authors: Shah Preeti , Katuri Varalakshmi , Awad Ola , Gul Naheed , Metzer Paul S. , Loeb David
Published: June 2012

Chordomas are slow growing, malignant cancers believed to originate from notochord remnants. Chordoma accounts for 1% of cancers of the central nervous system with an annual incidence of 0.08 cases per 100,000 individuals. There are 3 different histologic subtypes- classic chordoma, chondroid chordoma, and dedifferentiated chordoma. Chordomas are characterized by the presence of physaliphorous cells with vacuolated cytoplasm and high expression of the transcription factor T Brachury. Management of chordoma is primarily surgical – chemotherapy and radiation therapy have limited effect. The development of effective treatments for this tumor is hampered by the lack of adequate animal models. We report here the establishment of the first primary xenograft model of chordoma. Tumor excised from a patient diagnosed with classic chordoma was implanted adjacent to the right proximal tibia in a NOD/SCID/IL2Rγ (null) mouse. The resulting tumor has been serially transplanted at least 10 times with no diminution in growth rate over time. Histologically, the xenograft does not appear to be a classic chordoma; interestingly, the patient from whom the xenograft was derived relapsed with a dedifferentiated morphology. To confirm the identity of the tumor xenograft, T brachyury expression was demonstrated by immunohistochemistry and PCR. We have begun to characterize this xenograft by gene expression profiling and comparative genomic hybridization. Expression profiling results were verified by RT-PCR. Cells isolated from xenografts were analyzed by the Aldefluor assay to assess the levels of expression of aldehyde dehydrogenase activity. A cell line was generated from this xenograft as well and is being characterized. In summary, we report here the establishment of the first primary chordoma xenograft. Initial molecular characterization of the xenograft and a cell line derived from this tumor is ongoing. These tools will shed valuable light on the pathophysiology of this rare tumor and allow the preclinical development of novel treatment strategies.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2418. doi:10.1158/1538-7445.AM2011-2418
Abstract 2429: Increased incidence of brain metastases in BRCA1 related ovarian cancers
Authors: Sekine Masayuki , Tanaka Kenichi
Published: June 2012

Objective: Brain metastasis to ovarian cancer is a very rare phenomenon. BRCA1 related ovarian cancers show specific pathobiological profiles, an advanced stage, and sensitivity to chemotherapeutic agents. However, no clear relationship to any known metastatic behavior has yet been found, so we examined the BRCA1 mutation and expression profiles in ovarian cancer cases with brain metastases.

Methods: We examined our clinical records of 320 ovarian cancer cases from 1983 to 2008 in order to ascertain cases with brain metastases. In the molecular genetic analyses, we performed loss of heterozygosity (LOH), direct sequence, and immunohistochemical staining analysis of BRCA1. For LOH analysis, 4 markers spanning the region of BRCA1 were selected for use. The entire exons, 23 exons in BRCA1 and 26 exons in BRCA2, and the intronic boundary regions were sequenced in both forward and reverse directions for detecting germline mutations. The immunohistochemical studies were performed with the BRCA1 C-terminal mouse monoclonal antibody (Ab-3, Calbiochem,×500)

Results: We ascertained 7 cases with brain metastases in 320 ovarian cancer cases (7/320=2.2%). Among the 7 cases, 3 cases had ovarian and/or breast cancer patients in third-degree relatives. We detected 4 LOH-positive cases and a germline mutation of BRCA1 in 2 of the 4 cases, Q934X and L63X respectively. Furthermore, the remaining 2 cases showed an absent staining of the BRCA1 protein. Therefore, 4 out of 7 cases with brain metastases were considered BRCA1-related ovarian cancers (4/7=57.1%). All 4 of the cases were serous adenocarcinoma of ovary.

Conclusions: This is the first report of a hospital population-based study in relation to brain metastasis and BRCA1-related ovarian cancers. Our results suggest that the loss of BRCA1 function may be involved in the phenomenon of brain metastasis to ovarian cancer. Further molecular biologic analyses will be required for a better understanding of this rare phenomenon.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2429. doi:10.1158/1538-7445.AM2011-2429
Abstract 2431: Inhibition of metastasis through therapeutic targeting of focal adhesion kinase (FAK) in pancreatic cancer
Authors: Dhani Neesha C. , Schwock Joerg , Cao Ping-Jiang M. , Tsao Ming-Sound , Hedley David W.
Published: June 2012

BACKGROUND: Cancer mortality is primarily caused by metastatic disease; it has therefore been suggested that the preclinical evaluation of novel therapies should include testing in metastatic models of disease. This is especially relevant in light of recent reports of both conventional and targeted therapies being associated with accelerated metastases despite maintenance of primary tumor control. Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase which also acts as molecular scaffold, and localizes to focal adhesions, contact points between the cell and the extra-cellular matrix. Downstream of integrins and transmembrane receptors, FAK acts as a signaling hub, participating in processes integral to tumor progression including cell survival, proliferation, angiogenesis, migration and invasion. In this ongoing study, we explore the efficacy of selective genetic and pharmacologic FAK inhibition in preclinical models of human pancreatic cancer, including spontaneously metastasizing, cell-line derived and primary xenografts.

METHODS & RESULTS: We constructed modified pancreatic cancer cell lines using retroviral over-expression of the dominant negative FRNK (FAK related non-kinase). Pharmacologic inhibition of FAK was performed with PF-562271 (Pfizer), a selective FAK/Pyk2 inhibitor which recently completed Phase I testing. Adhesion, invasion and migration in vitro assays were conducted using the xCELLigence RTCA system. In vivo efficacy of FAK inhibition was evaluated in orthotopic xenograft models. In vitro studies demonstrated that both modalities of FAK inhibition resulted in impaired adhesion, invasion and migration and 3D colony formation, without comparable effects on viability, proliferation or colony formation in 2D. Expression of dominant-negative FRNK resulted in 49% smaller xenograft tumors (p=0.003), a 78% reduction in metastatic disease (p< 0.001) and longer overall survival (p < 0.001). There was a similar, 46% trend to reduction of metastatic burden (p=0.089) in orthotopic cell line xenograft models treated with PF-562271, despite a lack of significant primary tumor growth delay.

CONCLUSIONS: Our data suggest a potential utility of selective FAK inhibition in delaying metastatic progression of pancreatic cancer. This is particularly relevant clinically since patients frequently present with locally advanced, unresectable disease and subsequently develop metastases. Pharmacologic FAK inhibition (in combination with conventional therapies) is currently being evaluated in neo/adjuvant models of this disease.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2431. doi:10.1158/1538-7445.AM2011-2431