bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4146: Quantitative cell signaling analysis for cetuximab sensitivity prediction in colorectal cancer
Authors: Teppei Matsuo , Nishizuka Satoshi , Iwaya Takeshi , Ishida Kazushige , Noda Hironobu , Ikeda Miyuki , Kozai Megumi , Wakabayashi Go
Published: February 2011

[Background]

Anti-human EGFR monoclonal antibody (cetuximab) has been widely used in the treatment of colorectal cancer (CRC). However it is thought that a reliable predictor for therapeutic efficacy should be established because the benefit of cetuximab treatment is often still uncertain. Our preliminary study showed that the prediction of therapeutic efficacy using a conventional chemosensitivity test using growth suppression assay is generally not informative because cetuximab does not seem to have as strong a cytotoxicity as other anticancer drugs. KRAS mutation status has been used as a biomarker to predict therapeutic efficiency but it is not sensitive enough to determine the absolute indication.

[Purpose]

We hypothesized that we could predict therapeutic efficacy by evaluating expected molecular reaction when cetuximab is administrated in vitro. In the present study, we build up an in vitro EGFR signaling model for a new chemosensitivity test based on protein monitoring in response to cetuximab administration.

[Materials and Methods]

Three growth factors (EGF, TGF-α, and IGF) and the inhibitor (cetuximab) were added to HT29 (KRAS wild type, EGFR positive) in six combinations. Cell pellets were harvested at 5 time points over a 15min time course, each of which was processed for cell lysate. The lysates were analyzed by Western blotting to see if the proteins involved in the EGFR signaling pathway responded over time.

[Results]

We confirmed the activation of signaling stimulated by EGF, TGF-α, and IGF; and the effect on signaling by cetuximab in HT29 with Western blotting. Intriguingly, the phosphorylation level of EGFR was rapidly elevated and declined by EGF stimulating in the absence of cetuximab whereas dephosphorylation was prolonged in the presence of cetuximab. In the administration of either TGF-α or IGF, the phosphorylation pattern of EGFR, c-RAF, MEK, MAPK, ERK, PI3K, AKT, PTEN, and STAT was different from that of EGF alone.

[Conclusions]

These results shows that:(i) EGF is not EGFR's only ligand; (ii) cetuximab can't inhibit every signaling pathway starting from EGFR; and (iii) TGF-α and IGF activate signaling pathway starting from EGFR partially but not thoroughly. An additional reliable predictor of cetuximab efficacy should be identified based on functional protein characteristics.

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 4146.
Abstract 4147: Xanthohumol decreases Notch1 expression and cell growth in human epithelial ovarian cancer cell lines
Authors: Drenzek Jessica , Seiler Nicole , Rose Stephen
Published: February 2011

Epithelial ovarian cancer is the most lethal of all gynecologic malignancies in the United States with the majority of cases detected with advanced disease. Therefore, it is critical that novel therapies be developed that can effectively treat ovarian cancer. Notch1 is a transmembrane receptor that upon activation is cleaved by gamma-secretase resulting in Notch1 intracellular domain (NICD) translocation to the nucleus where it heterodimerizes with the transcription factor CBF-1 converting CBF-1 from a transcriptional repressor to a transcriptional activator via recruitment of coactivators. Deregulation of the Notch pathway has been observed in cancers such as T cell lymphoblastic leukemia, pancreatic, and breast. In addition, it has been shown that NICD is expressed in ovarian cancer cell lines and ovarian tumors. Xanthohumol, a prenylated chalcone derived from hops used in brewing beer, has been shown to exhibit anti-cancer properties. Since xanthohumol has been shown to inhibit cancer cell growth and invasion, we hypothesized that the Notch1 signaling pathway is suppressed by xanthohumol leading to decreased cell growth. OVCAR3 and SKOV3 are human epithelial ovarian cancer cell lines that express high levels of NICD. These cells were treated with increasing doses of xanthohumol, and cell growth was measured by 3-[4,5-dimethylthiazole-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay on days 0, 2, 4, and 6. In addition, protein lysates were isolated on days 2 and 4 of treatment, detected by western blot using a NICD primary antibody, and protein expression was evaluated by densitometry. These treatments resulted in decreased cell growth when measured by MTT assay. In addition, when analyzed by western blot, there was a decrease in NICD protein expression. In conclusion, xanthohumol was a potent inhibitor of ovarian cancer cell growth, and our results suggest that xanthohumol is affecting the Notch pathway. Therefore, it is essential that further investigation into the role that xanthohumol has on the Notch pathway be pursued. These outcomes could prompt the use of xanthohumol as a potential chemotherapeutic agent in ovarian cancer patients who do not respond long term to standard chemotherapy.

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 4147.
Abstract 4151: Mutual feedback activation of PI3K and MAPK pathways support combining inhibitors of the two pathways to enhance efficacy
Authors: Zhang Theresa , Feldman Igor , Paweletz Cloud , Nebozhyn Michael , Shumway Stuart Shumway , Chen Albert Chen , Roberts Brian Roberts , Arthur William , Sathyanarayanan Sriram , Lutterbach Bart , Strack Peter , Huang Pearl , Dai Hongyue , Loboda Andrey , Watters James
Published: February 2011

There has been increasing interest in developing cancer therapies targeting PI3K pathway nodes. Unfortunately, the number of tumor types responding to PI3K pathway inhibitors as monotherapy has been limited, since most cancer cells accumulate genetic alterations that result in the constitutive activation of a complex network of proliferation and survival signals. As such, the inhibition of a single survival/proliferation pathway or node within this broader network may be circumvented by constitutive activation (or feedback activation) of an alternative survival pathway, and thus be insufficient for inducing a clinical response. Therefore, in most tumor types, dual pathway inhibition, guided by an in-depth understanding of feedback and cross-talk between pathways, may be required to induce tumor regression. Here we present a nonclinical study exploring mutual feedback activation of MAPK and PI3K pathways. Inhibition of the PI3K pathway by MK-2206 (AKT inhibitor) or MK-8669 (mTOR inhibitor) resulted in feedback activation of MAPK pathway nodes in subsets of lung cell lines. Similarly, inhibition of the MAPK pathway by a MEK inhibitor resulted in feedback activation of the PI3K pathway. Notably, a lack of such feedback activation was associated with sensitivity to the mono inhibitors. The strength of the feedback activation correlated with changes of several receptor tyrosine kinases, suggesting them as a part of the feedback mechanism. Finally, combined inhibition of both MAPK and PI3K pathways synergistically inhibited the feedback activation of both pathways and led to improved in vitro efficacy. Taken together, our findings identify mutual feedback activation of the PI3K and MAPK pathways in response to specific inhibitors and underscore the importance of combined therapeutic approaches with inhibitors to both pathways.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4151.
Abstract 4154: Intratumoral heterogeneity of HER2/neu expression in diffuse-gastric carcinoma of European patients and its relevance for patients’ management
Authors: Tomasic Gorana , Malouf Gabriel G. , Marty Virginie , Malka David , Vielh Philippe , Ducreux Michel , Boige Valerie
Published: February 2011

Background: This study assessed the human epidermal growth factor receptor-2 (HER2) protein expression, its relationship with gene amplification and its intra-tumoral heterogeneity in diffuse-gastric carcinoma, a subset of gastric carcinoma that might benefit from Trastuzumab in case of HER2 amplification according to recently reported ToGA trial (J Clin Oncol 27:18s, 2009 (suppl; abstr LBA4509)

Patients and methods: The study included 51 European consecutive patients who underwent surgical resection in our institution between January 1991 and December 2007 for diffuse gastric carcinoma. A tissue-microarray was constructed using paraffin-embedded tissues of gastrectomy and matched lymph nodes. HER2 were evaluated by immunohistochemistry (IHC), using the same criteria than for breast cancer. All samples with a 2+ or 3+ HER2 overexpression were evaluated by FISH.

Results: HER2 overexpression was observed in 2 (4%) tumors and there was intratumoral heterogeneity. The first tumour shows 2+/3+ immunostaining in lymph nodes with negative immunostaining in the primary tumor. The second tumor shows a focal 3+ HER2 immunostaining in the primary tumor without evidence of positive immunostaining in lymph nodes and peritoneal metastasis. Using FISH, HER2 3+ tumour areas had a gene amplification, whereas no amplification was found in 2+ tumor. The 2 HER2 positive tumors showed the same heterogeneous pattern, with adjacent 3+ positive and negative areas by both IHC and FISH. There was a 100% concordance between TMA and the tissue-sections. Despite complete resection, the 2 patients with HER2 expression died within 15 months.

Conclusions: This study showed that 4% of resected diffuse gastric carcinoma had a heterogeneous distribution of HER2 gene amplification. These findings raise the question whether HER2 testing should be realized systematically in these patients and whether Trastuzumab may benefit in tumours with heterogeneous HER2 expression.

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 4154.
Abstract 4155: The small leucine-rich proteoglycan biglycan localizes to the nucleus of neoplastic cells in glioblastoma biopsies and tumor xenografts
Authors: deCarvalho Ana C. , Berezovsky Artem , Lemke Nancy , Mikkelsen Tom , Kalkanis Steven N.
Published: February 2011

Global molecular profiling efforts are leading to the identification of prognostic gene expression signatures for gliobastoma. Recently, several extracellular matrix (ECM) components typically expressed in mesenchymal tissues, have been shown to be overexpressed in a subgroup of high grade gliomas from poor prognosis patients, including the highly related small leucine-rich repeat proteoglycans (SLRPs) biglycan and decorin. These proteins were originally isolated from cartilage and are widely expressed in differentiated mesenchymal tissues, but their involvement in glioblastoma pathology is not understood. Biglycan expression in glioblastoma surgical specimens, cancer stem cells, and xenografts was assessed by RNA microarray, real-time PCR, Western blot and immunohistochemistry, and compared to decorin. Over 80% of the glioblastoma surgical specimens expressed both proteoglycans in the pericellular location of endothelial cells. In neoplastic cells, while decorin was observed in the pericellular location of discrete regions of about 40% of the samples, biglycan localized to the nucleus in most tumors analyzed. It has been previously reported that biglycan possess a nuclear localization signal, but in vivo nuclear localization in a glial tumor has not been shown. The unglycosylated core biglycan protein was ubiquitous, while the glycosylated form was less uniformly distributed among the tumor samples. Glioblastoma cancer stem cell enriched neurosphere cultures derived from the tumor specimens did not express biglycan or decorin in vitro. However, biglycan was re-expressed upon tumor formation, as nuclear staining was observed in the orthotopic tumor xenograft initiated by the neurospheres. Using the Rembrandt data base (http://caintegrator-info.nci.nih.gov/rembrandt) we observed a robust inverse correlation of biglycan expression in GBMs with survival. The nuclear localization in GBMs suggests an unconventional role for this proteoglycan in the pathology of this malignancy.

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 4155.
Abstract 4160: Evaluating epithelial-to-mesenchymal transition (EMT) in the EL1-luc/EL1-SV40 T-antigen transgenic mouse model of pancreatic cancer
Authors: Franklin Maryland Rosenfeld , Krueger Joseph , Young G. David , Sennello Regina , Leary Lisa , Zhong Shaobin , Chen Hao , Lim Ed , Zhang Ning , Lassota Peter , Wild Robert
Published: February 2011

Epithelial-to-mesenchymal transition (EMT) is a biological function important in normal cellular processes such as embryonic development and wound healing. In cancer it is thought that the tumor cell machinery can re-activate these normal pathways resulting in more aggressive and invasive tumors. The loss of E-cadherin and the gain of vimentin are hallmarks which identify the process of EMT and have been shown to correlate with poor prognosis in multiple solid tumor types. While many preclinical models are utilized to evaluate mechanisms of tumorigenesis few in vivo models evaluating parameters of EMT have been described.

The EL1-luc/EL1-SV40 T-antigen transgenic mouse represents a model of pancreatic cancer whereby mice develop tissue specific, spontaneous and bioluminescent pancreatic tumors. To evaluate whether EMT occurs in the EL1-luc/EL1-SV40 T-antigen model in vivo, we collected primary pancreatic tissue from male mice between 10 and 21 weeks of age. The tissue was formalin fixed, paraffin embedded and then utilized for histopathological endpoints such as Hemotoxylin and Eosin staining as well as immunohistochemistry for markers known to be involved in EMT such as E-cadherin and vimentin. We found the tumors to express both markers and become very heterogeneous over time. In early tumors E-cadherin expression is membrane localized and very high. Over time there are areas of the tumors that have reduced or lost E-cadherin expression. Vimentin expression was highly variable but when present tended to be highly expressed. In many of the later stage tumors there was substantial heterogeneity reflected by the appearance of multiple cell types within a tumor. We utilized the Aperio (Aperio Technologies, Vista, CA) slide scanner and software system to evaluate serial sections of tumor samples and found that in some sections of the tumor E-cadherin is present and vimentin is absent, whereas in other areas of the tumor vimentin is present in the absence of E-cadherin. Additionally, we identified areas of the tumor that seem to be expressing both markers which suggests that the EL1-luc/EL1-SV40 T-antigen transgenic mouse may recapitulate many aspects of EMT observed in vivo, thus offering a model system to study the signaling and molecular changes necessary for this process during cancer progression.

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 4160.
Abstract 4161: Synergistic effects of aflatoxin B1 exposure and glycine N-methyltransferase deficiency in hepatocarcinogenesis
Authors: Liu Shih-Ping , Li Ying-Shiuan , Lee Cheng-Ming , Yen Chia-Hung , Liao Yi-Jen , Huang Shiu-Feng , Chien Chau-Heng , Chen Yi-Ming Arthur
Published: February 2011

In both humans and rodents, males are known to be more susceptible than females to hepatocarcinogenesis. We have previously reported that glycine N-methyltransferase (GNMT) interacts with aflatoxin B1 (AFB1) and reduces both AFB1-DNA adduct formation and hepatocellular carcinoma (HCC) in mice. We also reported that 50% of the males and 100% of the females in a small group of Gnmt null (Gnmt−/−) mice developed HCC, with first liver nodules detected at mean ages of 17 and 16.5 months, respectively. In this study we tested our hypothesis that male and female Gnmt−/− mice are susceptible to AFB1 carcinogenesis, and that the absence of Gnmt expression may accelerate AFB1-induced liver tumorigenesis. We inoculated Gnmt−/− and wild-type mice intraperitoneally with AFB1 at 7 days and 9 weeks of age and periodically examined them using ultrasound. Liver nodules were detected in 6 of 8 males and 5 of 5 females at 12.7 and 12 months of ages, respectively. Liver nodules from 5/8 (62.5%) male and 4/5 (80%) female Gnmt−/− mice were diagnosed as having HCC, approximately 6 months earlier than AFB1-treated wild-type mice. Results from microarray and real-time PCR analyses indicate that five detoxification pathway-related genes were down-regulated in AFB1-treated Gnmt−/− mice: Cyp1a2, Cyp3d44, Cyp2d22, Gsta1 and Abca8a. In summary, we observed a synergistic relationship between GNMT deficiency and AFB1 exposure in hepatocarcinogenesis, further evidence that GNMT over-expression is an important contributing factor to female liver cancer resistance.

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 4161.
Abstract 417: Tax-interacting protein 1 (TIP-1) as one biomarker of invasive astrocytomas
Authors: Wang Hailun , Hallahan Dennis , Han Zhaozhong
Published: February 2011

Despite the comparatively low incidence of astrocytomas to other human cancers, mortality rates of cancer patients with invasive astrocytomas are extremely high. Despite decades of therapeutic research, curative intervention is still nonexistent for high grade astrocytomas; patient care ultimately focuses on palliative management. Even with aggressive surgical, radio- and chemo- therapies, highly invasive astrocytomas have poor prognosis with an average survival time of 12 months. We intend to identify and characterize new biomarkers or molecular targets for effective diagnostics or therapeutics development. In our previous study of recombinant peptide for tumor imaging, we found that tumor xenografts formed with D54 glioma cells had relatively higher binding to HVGGSSV peptide than other tumor models. It was further elucidated that Tax-interacting protein 1 (TIP-1) contributes to the HVGGSSV peptide binding within tumors. Immunohistochemistry study of human brain tissues revealed significantly elevated expression of TIP-1 within astrocytomas, while compared to normal brain tissue or oligodendrogliomas that showed faint to negative staining of TIP-1. More interestingly, the highest expression of TIP-1 was found in glioblastoma multiforme, the highly invasive astrocytomas. To elucidate biological relevance of TIP-1 expression in invasive astrocytomas, human glioma cell line D54 was genetically engineered to have elevated or down-regulated expression of TIP-1. Tumorigenesis studies indicated that TIP-1 positively regulated tumor formation in nude mice. Knocking-down TIP-1 within D54 cells resulted in delayed tumor formation and decreased tumor growth rate. However, over-expression of TIP-1 within D54 cells lead to fast growing tumors in vivo, histological examination showed the TIP-1 over-expressing D54 tumors were sporadic with presence of necrotizing tissues surrounded by anaplatic cells, one characteristic of glioblastomas multiforme. In vitro characterization further demonstrated that the TIP-1 over-expressing cells had improved invasion capability, TIP-1 is dominantly expressed in invasion front of D54 cells. Biochemical studies indicated that the TIP-1 modulates cytoskeleton organization by interacting with multiple proteins. In summary, this study demonstrated that TIP-1 is one biomarker of invasive astrocytomas, and might be one potent molecular target for therapeutics development.

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 417.
Abstract 4173: In vivo and in vitro propagation of Intraductal Papillay Mucinous Neoplasms
Authors: Kamiyama Hirohiko , Kamiyama Mihoko , Hong Seung-Mo , Karikari Collins , Lin Ming-Tseh , Griffith Margaret , Young Angela , Kirby Alexis , Borges Michael , Lubek Conrad , Mizuma Masamichi , Feldman Georg , Shi Chanjuan , Liang Hong , Goggins Michael , Maitra Anirban , Hruban Ralph , Eshleman James
Published: February 2011

Background

Intraductal Papillary Mucinous Neoplasms (IPMNs) are one of the 3 known curable precursor lesions (IPMN, MCN (Mucinous Cystic Neoplasm), and PanIN (Pancreatic Intraepithelial Neoplasm)) of invasive pancreatic ductal adenocarcinoma, an almost uniformly fatal disease.

Methods

To advance the study of the biological features of IPMNs, we attempted in vivo and in vitro growth of selected IPMNs, based on the hypothesis that IPMNs could be grown in the most severely immunodeficient mice. We examined fourteen cases by implanting them into nude, severe combined immunodeficient (SCID), and NOD/SCID/IL2Rγnull (NOG) mice, in addition to direct culture, to generate tumor xenografts and cell lines.

Results

Thirteen tumors were implanted into the 3 types of mice, including 10 tumors implanted into the triple immunodeficient NOG mice, where the majority (8 of 10) grew including 5 IPMNs lacking invasive components. One of the explanted IPMNs, with an associated invasive carcinoma, was successfully established as a cell line. Growth in soft agar, growth in mice and homozygous deletion of P16/cdkn2a confirmed its tumorigenicity. The expression of cytokeratin confirmed the epithelial differentiation of the cell line, and DNA fingerprinting confirmed its human origin.

Future directions

Isolating cell lines from IPMNs and their invasion counterparts should be valuable to identify gene mutations critical to IPMN carcinogenesis, and permit drug screening to identify compounds that cause regression of those lesions.

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 4173.
Abstract 4180: The L360R point mutation in the DNA repair gene XRCC1 suppresses tumor progression
Authors: Pettan-Brewer Christina , Goh Jorming , Coil Rebecca , Enns Linda , Morton John , Ladiges Warren
Published: February 2011

Variations in the DNA repair gene X-ray repair cross-complementing group 1 (XRCC1) have been increasingly reported in cancer epidemiology investigations, but have yielded conflicting results and are lacking biological data. XRCC1 is an essential scaffolding protein that interacts with many proteins associated with base excision repair and single-strand break repair. Several polymorphisms and point mutations have been shown to be in the BRCT1 binding domain, where critical binding with the DNA damage sensor gene Poly (ADP-ribose) polymerase (PARP) occurs. The point mutation L360R interferes with PARP binding in cell culture systems. In order to further investigate the biological effects of L360R, we constructed a gene-targeted mouse line and showed the homozygous genotype to be embryonic lethal. We therefore used the heterozygous genotype to determine tumor susceptibility and showed that subcutaneously implanted B16 melanoma tumors developed more slowly in L360R mutant mice compared to wild type littermates (tumor volume 116±11 mm3 vs 263±21 mm3, respectively, p≤0.05) suggesting host mediated affects. We conducted a second experiment using azoxymethane (10mg/kg once a week for 6 weeks) to induce colon tumors and showed that after six months the tumor burden was 14.2±3 mm3 in mutants compared to 22.5±5 mm3 in wild type littermates (p≤ 0.05). These findings suggest that the XRCC1 L360R point mutation acts as a tumor suppressor, possibly through host mediated events. The molecular mechanism for the suppression is not yet known, but the XRCC1 L360R mouse line is a promising animal model to further investigate and determine the clinical relevance of this novel observation.

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 4180.
Abstract 4186: Development of a novel, genetically faithful model of pediatric glioblastoma
Authors: Ihrie Rebecca A. , Huillard Emmanuelle , Petritsch Claudia , James David , McMahon Martin , Gutmann David H. , Rowitch David H. , Alvarez-Buylla Arturo
Published: February 2011

Gliomas are among the most lethal human cancers, with high-grade tumors exhibiting a dismal 5-year survival rate. Mutations and amplifications of receptor tyrosine kinases such as PDGFR and EGFR, which drive Ras-MAPK pathway activity, are among the hallmark mutations of adult gliomas. However, pediatric glioblastomas often do not exhibit the oncogenic mutations found in adult tumors, suggesting that these tumors may have distinct mechanistic origins. The unique genetic characteristics of pediatric brain tumors may also affect their response to conventional treatment. Therefore, there is a need to generate mouse models that faithfully recapitulate the genetics of pediatric glioblastomas to identify potential avenues for improving treatment of these tumors. Recent genetic profiling of pediatric brain tumors has found activating mutations of the BRaf kinase in higher grade tumors but not low-grade juvenile pilocytic astrocytomas. By contrast, BRaf is not frequently altered in adult gliomas, suggesting that cells in the developing brain may be particularly susceptible to this mechanism of Ras pathway activation. In addition, BRaf mutation is closely linked to the loss of the tumor suppressor Ink4a/Arf, indicating a possible requirement for loss of this tumor suppressor in the development of high-grade gliomas.

Here, we describe the generation of a model system allowing expression of oncogenic BRaf in spatially and temporally defined populations of neural stem cells. In BRaf CA/+; R26YFP animals, Cre-mediated recombination induces expression of the oncogenic V600E mutant form of Braf from the endogenous gene locus and simultaneously labels these cells via expression of YFP. By stereotaxically injecting Cre-expressing adenovirus into the neonatal or adult brain in these mice, we can induce oncogene expression in specific populations of neural progenitors and track its effects on infected cells.

Data from multiple tumor types suggests that the activation of Ras-MAPK signaling via BRaf mutation is tightly linked to loss of the Ink4a/Arf tumor suppressor locus. We are also using viral introduction of Cre to activate oncogenic BRaf when Ink4a/Arf expression is absent. The phenotype of these mice will be compared to human pediatric gliomas to determine if this system provides an accurate model of the human disease. Such a model would provide a platform for testing targeted therapies against BRaf - a critical step in designing new treatments for pediatric glioblastoma.

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 4186.
Abstract 4193: Treatment of malignant gliomas with systemic rapamycin and convection-enhanced delivery of nanoliposomal CPT-11
Authors: Marina Mendiburu-Eliçabe , Hadaczek Piotr , Forsayeth John , Bankiewicz Krystof
Published: February 2011

Combined therapy of rapamycin and nanoliposomal irinotecan (nLs-CPT-11) was used in the treatment of U87-MG intracranial brain tumor model looking for an improvement in the survival in comparison to animals that received only single-agent therapy.

The prognosis for patients with malignant gliomas has remained dismal, and the median survival for patients with glioblastomas is still less than one year after prognosis despite multidisciplinary use of surgery, chemotherapy and radiotherapy. Combination of two agents had start to be an encouraging option to treat glioblastomas giving better results when two different molecules implicate in tumor progress are inhibited instead than only one. Clinical trials with convection-enhanced delivery (CED) have opened a new window in neuro-oncology to the direct delivery of chemotherapeutics to the central nervous system (CNS), circumventing the blood-brain barrier (BBB) and reducing side effects. In this study, we evaluated a combination of nLs-CPT-11 and rapamycin to enhance efficacy in brain tumor xenograft models, and to establish a combination treatment (systemic rapamycin and CED nLs-CPT-11) capable of improving survival from malignant brain tumors. CPT-11 suppress activity of a key enzyme involved in tumor cell replication, topoisomerase I. Rapamycin inhibits PI3K/Akt/mTOR pathway implied in the regulation of the cell proliferation, cell growth, differentiation, migration and survival. In vitro toxicity experiments were carried out with U87-MG and U251-MG cells. An in vivo survival study was performed for intracranial brain tumor xenograft. The combination displayed excellent efficacy in a survival study 7 days after tumor implantation. Animals in the control group had a median survival of less than 20 days, whereas the combination group triplicates the median survival, surviving at least 70 days all the animals. We conclude that CED of nLs-CPT-11 and systemic rapamycin may be an effective treatment option for malignant gliomas.

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 4193.
Abstract 4199: A transgenic mouse for imaging caspase-dependent apoptosis within the skin
Authors: Hamilton Chritin A. , Khanna Divya , Bhojani Mahaveer S. , Lee Kuei C. , Dludosz Andrej , Ross Brian D. , Rehemtulla Alnawaz
Published: February 2011

Apoptosis is an essential process for the maintenance of normal physiology. The ability to non-invasively image apoptosis in living animals would provide unique insights into its role in normal and disease processes. Herein, a recombinant reporter consisting of LacZ gene flanked by two ER regulatory domains and intervening DEVD sequences was constructed to serve as a tool for invivo assessment of apoptotic activity. The results demonstrate that when expressed in its intact form, the hybrid reporter had undetectable β-galactosidase activity. Caspase-3 activation in response to an apoptotic stimulus resulted in cleavage of the reporter, and thereby reconstitution of β-galactosidase activity. Enzymatic activation of the reporter during an apoptotic event enabled non-invasive measurement of β-galactosidase activity in living cells, which correlated with traditional measures of apoptosis in a dose and time dependent manner. Furthermore, using a near-infrared fluorescent substrate of β-galactosidase (DDAOG), non-invasive in vivo imaging of apoptosis was achieved using a xenograft tumor model in response to pro-apoptotic therapy. Lastly, a transgenic mouse model was developed expressing the ER-LacZ-ER reporter within the skin. This reporter and transgenic mouse could serve as a unique tool for the study of apoptosis in living cells and animals especially in context of skin biology.

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 4199.
Abstract 420: Metastatic and neo-vasculature determinants as prognostic markers of thyroid cancer
Authors: Rajoria Shilpi , Suriano Robert , Shanmugam Arulkumaran , Megwalu Uchechukwu , Schantz Stimson P. , Geliebter Jan , Tiwari Raj K.
Published: February 2011

Thyroid is amongst one of the most vascularized tissues of the human body and studies have demonstrated that neo-vascularization is critical for growth and metastasis of thyroid carcinoma with well differentiated thyroid carcinoma metastasizing to the lymph nodes of the neck in 50% of the patients. The perceived risk of thyroid proliferative diseases in women is one in eight, a risk similar to that of breast cancer, suggesting a possible role of female reproductive hormones such as estrogen in this epidemiological distribution. The identification of systemic modulators such as estrogen and markers of metastatic phenotype or neo-vascularization in thyroid cancer is a clinical need. We developed an in vitro thyroid cell culture model to study the effect of estrogen on metastatic phenotype of thyroid cells. We discovered that estradiol modulates metastatic properties of these thyroid cells as evidenced by an increase in in vitro migration, invasion and adhesion in presence of estradiol which correlates with a concomitant downregution of tumor suppressive protein, β-catenin. Restitution of cadherin/catenin complex by fulvestrant suppresses the metastatic phenotype of thyroid cells thus validating our findings of β-catenin as a significant metastatic phenotype marker. Furthermore, we developed an in vivo animal model so as to study the effect of estrogen on neo-vasculature in thyroid cancer. We found that estradiol acts as a mobilizing agent for bone marrow derived endothelial progenitor cells (BM-EPCs) resulting in homing of enhanced numbers of BM-EPCs to orthotopic tumor when ovariectomized mice were supplemented with estradiol pellets. We also discovered that thyroid cells secrete paracrine factors which results in enhanced migration of BM-EPCs as evidenced by in vitro experimental model suggesting that mobilization of BM-EPCs is tumor responsive. In conclusion, our data provides strong evidence that estradiol enhances the metastatic propensity of thyroid cells and mobilizes BM-EPCs to the thyroid tumor. These findings will allow for the possible development of anti-estrogenic compounds that target catenin mediated metastasis and/or stem cells mediated neo-vascularization of thyroid 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 420.
Abstract 4203: Plasma and tumor pharmacokinetics (PK) of carboplatin in Genetically Engineered Mouse Models of melanoma (GEMMs), murine melanoma, and in patients with cutaneous melanoma
Authors: Combest Austin J. , Roberts Patrick J. , Dillon Patrick J. , Habibi Sohrab , Eiseman Julie L. , Strychor Sandra , Hanna Suzan K. , Muller Markus , Brunner Matthias , Ross Charlene M. , Sharpless Norman E. , Zamboni Willam C.
Published: February 2011

Background: It is currently unclear what preclinical tumor model most accurately reflects the tumor disposition and antitumor response of an anticancer agent in patients with solid tumors. GEMMs of melanoma and other solid tumors may better predict drug distribution and antitumor response compared with flank tumor models by allowing for the development of tumor in situ which may more faithfully recapitulate human cancer. We compared the plasma and tumor disposition of carboplatin in a GEMM and murine models of melanoma and in patients with cutaneous melanoma.

Methods: Carboplatin was administered at 50mg/kg IV x 1 via a tail vein to a GEMM of melanoma [tyrosinase-H-RasG12V Ink4a/Arf−/− (TRIA)] and mice bearing B16 murine melanoma. Plasma total and ultrafiltrate conc were obtained (n = 3 mice per time point) from 0 to 6 h after administration. Microdialysis studies evaluating carboplatin in tumor extracellular fluid (ECF) were performed (n = 6 mice) from 0 to 4 h after administration. Six patients with cutaneous metastatic melanoma were administered carboplatin at 400mg/m2 IV over 20 minutes. Plasma total and tumor ECF, collected by microdialysis, were obtained from 0 to 6 h after administration. Platinum (Pt) conc were measured by ICP-MS, FAAS and FAAS in the GEMM, B16 and patient studies, respectively. Areas under the plasma conc versus time curves from 0 to infinity (AUC) of standard and allometrically scaled data were calculated.

Results: Results are in the table: UnitsHumanGEMMB16Standard / Unscaled    Plasma Total Pt AUCµg/mL·h26.028.540.5Plasma Ultrafiltrate Pt AUCµg/mL·h—25.427.9Tumor ECF Pt AUCµg/mL·h15.38.61.9Allometrically Scaled    Plasma Total Pt AUC(µg/mL)/(mg/kg)]·(h/kg)0.841.372.15Tumor ECF Pt AUC(µg/mL)/(mg/kg)]·(h/kg)0.490.410.10Ratio of Plasma Total pt AUC to Tumor ECF pt AUC 0.580.300.05

Conclusion: The tumor PK of carboplatin in GEMM models of melanoma more closely resembles the tumor disposition in patients with melanoma as compared with flank tumor models. GEMMs show promise in becoming an improved prediction model for drug PK and response in patient tumors. Studies are being conducted to determine the basis for differences in tumor drug delivery between GEMMs and transplanted tumors.

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 4203.
Abstract 4209: Organotypic multicellular glioma spheroids prepared from tissue fragments obtained by ultrasonic aspiration
Authors: Jensen Stine S. , Aaberg-Jessen Charlotte , Jabbari Javad , Andersen Claus , Schrøder Henrik D. , Kristensen Bjarne W.
Published: February 2011

Ultrasonic aspirators are commonly used to resect brain tumors because they allow safe, rapid and accurate removal of dissected tissue. However, the tissue fragments removed by ultrasonic aspirators are used surprisingly little in daily diagnostics and in clinical research. Due to the increasing need for vital human tumor tissue for these purposes, which today require multidisciplinary approaches, the aim of this study was to investigate whether tissue fragments obtained by ultrasonic aspiration could be cultured as organotypic multicellular spheroids. These spheroids have earlier been shown to represent an important alternative to commercially available cell lines having multiple mutations and phenotypic changes. Fragments obtained by ultrasonic aspiration (sonocut) from one anaplastic astrocytoma and seven glioblastomas and tumor biopsy tissue from four of these glioblastomas were included in the present study. Sonocut and tumor biopsy tissue were mechanically dissociated into small tissue fragments and cultured in a serum containing medium on agar-coated plates. If excess tissue was available the fragments were also cultured in a serum-free medium. After 12-18 days of culturing the tissue fragments had formed spheroids which were fixated and paraffin embedded. Hematoxylin eosin staining was performed in order to investigate the viability and morphology of the spheroids. Part of the material was stained immunohistochemically for the astrocytic marker GFAP and the blood vessel markers VWF, CD31 and CD34. In the present study all sonocut cultures contained viable spheroids, whereas one of the cultures obtained from tumor biopsy tissue did not. The spheroids were able to survive and proliferate under both serum and serum-free conditions. The spheroids from both sonocut and tumor biopsy tissue contained malignant traits including high cellularity, cellular pleomorphism, mitoses and necrosis. Some spheroids consisted solely of GFAP positive tumor cells, while others contained preserved blood vessels confirmed by VWF, CD31 and CD34 immunohistochemistry. In conclusion, we found that it was possible to obtain viable spheroids from sonocut-material under both serum and serum-free conditions.

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 4209.
Abstract 4221: Pregnancy associated mammary gland micro-chimerism indicates fetal progenitor integration in breast cancer
Authors: Aziz-Zaman Sonya , Denes Viktoria , Pilichowska Monika , Geck Peter
Published: February 2011

The PURPOSE of this work was to use Y chromosome markers to investigate the hypothesis that (i) post-pregnancy mammary glands are chimeras that integrate stem cells from the fetus and (ii) these immature progenitors have a critical role in breast cancer.

RATIONALE The exchange of fetal and maternal cells during pregnancy results in the integration of fetal, multipotent progenitors in the mother. The established fetomaternal microchimerism (MC) appears to be stable for life in most mothers. These cells, called Pregnancy Associated Progenitor Cells (PAPC) represent a surprising, third type of progenitor pool (in addition to embryonic- and adult stem cells), with major biological implications. Y chromosome markers (male fetus) detect the fetal lineage and were present in several maternal tissues. No data are available on fetal progenitors in the human mammary gland, although the hormonal regeneration-degeneration cycles render the mammary gland a prime target for the integration of circulating stem/progenitor cell.

HYPOTHESIS Based on the above, we postulated that the fetal progenitor lineage is highly represented in the mammary gland with a critical role in breast cancer. Two biological outcomes were considered: (i) PAPC integration may correctly rejuvenate the stem cell pool and protects from breast cancer; (ii) differentiation programs in the fetal progenitor lineages fail and progress to breast cancer. Our major OBJECTIVE was to investigate if breast cancer is microchimerism related by using Y-chromosome markers in breast cancer samples to establish correlations with breast cancer.

EXPERIMENTAL PROCEDURES We worked out the methodology for Y-marker detection in breast cancer samples from commercially available tissue-arrays. Methods for microdissection (laser and mechanical), DNA extraction and PCR for Y-specific sequences were optimized. We targeted two Y-specific genes: SRY (SOX3-unrelated regions) and DYS14 (a male marker for a TSPY pseudogene). We analyzed 65 samples mostly from invasive ductal breast carcinomas and 40 normal controls.

SUMMARY OF RESULTS We found low level Y-representation in 17.5 % (7/40) of normal breast samples. We detected increased levels of Y markers in 33.8 % (22/65) of breast cancer tissues (integrated SRY and DYS14 data). Further studies to analyze clinical correlations are in progress.

CONCLUSIONS The data suggest, for the first time, that fetal cells are able to integrate into the mammary gland. Furthermore, this integration event appears to coincide with breast cancer. The increased representation suggests potential involvement of fetal progenitor cells in the pathomechanisms of 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 4221.
Abstract 4222: CCN5 deficient MCF-7 side population exhibits stem cell like behavior and formed aggressive tumor in xenograft model
Authors: Dhar Kakali , Banerjee Snigdha , Slusser Joyce , Majumder Monami , Banerjee Sushanta K.
Published: February 2011

The side population (SP) has been identified in a variety of solid tumor cell lines including human breast cancer cell lines. This population of cells has been considered as cancer stem cells (CSCs). Cancer recurrence is currently thought to be due to non-dividing cancer stem/progenitor cells that are resistant to chemo- and radio-therapies. Different therapeutic approaches need to be considered for the elimination/transformation of the cancer stem cells. To reshape our therapeutic approach to breast cancer, we aimed this study and isolated the SP from MCF-7 breast cancer cell line. SP was confirmed by both positive and negative cell surface molecular markers. These include CD44, CD24, b-catenin, E-cadherin, Keratin 19, Oct-4, Notch-1 and Vimentin. Our studies showed that SP and non-SP exhibit differential expression of CCN5, an anti-invasive gene. CCN5 expression was undetected or barely expressed in SP. The comparative studies showed that the in vitro migratory property of SP was significantly higher as compared to non-SP. Moreover, the introduction of CCN5 in SP may enhance the epithelial properties as evidenced by differential expression of MET (mesenchymal-epithelial transition) molecular markers. SP shows higher tumorigenicity in immunodeficient mice. These findings indicate that CCN5 deficiency could be an essential molecular event for the development of a side population having invasive phenotype. Therefore, CCN5 can be used as a therapeutic reagent for advanced 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 4222.