bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 323: Use of triple combination array analysis to identify STEAP family member 4 (STEAP4), a novel tumor suppressor gene in hepatocellular carcinoma
Authors: Nomoto Shuji , Okamura Yukiyasu , Hayashi Masamichi , Takeda Shin , Sugimoto Hiroyuki , Fujii Tsutomu , Nakao Akimasa
Published: June 2012

Background: Hepatocellular carcinoma (HCC) is one of the most common cancers world-wide, but the molecular mechanisms underlying hepatocarcinogenesis are not clear. Array technology has made it possible to identify novel genes with alterations in liver cancer. In this study, we performed triple combination array analysis, expression profiling, karyotyping analysis using SNP array, and array-based DNA methylation profiling in the same HCC sample and attempted to find a novel tumor suppressor gene as a prognostic marker.

Materials & Methods: A 68-year-old woman with chronic hepatitis C was diagnosed with HCC. The tumor and corresponding normal tissues were used for extraction of DNA and RNA. SNP chip array experiments were performed according to the standard protocol of Affymetrix GeneChip Mapping 500K arrays for DNA. mRNA expression profiles were determined using Affimetrix HGU133A and B Gene-Chips. Bisulphite-converted genomic DNA was analyzed using Illumina's Infinium Human Methylation27 Beadchip Kit. We consequently analyzed the identified gene using quantitative RT-PCR, methylation and un-methylation-specific PCR in nine HCC cell lines and in 48 primary HCC tissues and corresponding non-cancerous tissues collected at Nagoya University Hospital, and then compared these results with clinico-pathological data.

Results: The STEAP4 gene, is located on 7q21.12, and expression was decreased at a Log2 ratio of -4.2 to -2.0 in cancerous tissue. There were two copies of 7q21.12, indicating no loss of heterozygosity (LOH) in the SNP array. The methylation value (0-1.0) in the tumor sample was 0.870 compared to 0.234 in non-cancerous tissue, indicating high methylation in tumor tissue in the Methylation array. Re-activation of STEAP4 expression was seen after 5-aza-dC treatment in HCC cell lines. Moreover, we found that 32 of 48 (67%) HCC samples showed promoter hypermethylation. In 32 methylated cases, the expression values of STEAP4 gene in tumor tissues by real-time RT-PCR were tended to decrease compared to corresponding normal samples. In addition, methylated cases were significantly correlated with positive results for Hepatitis B and α-feto protein and had a tendency for poor prognosis (p=0.098).

Conclusion: The STEAP4 gene is a tumor suppressor gene in HCC and might be a marker of poor prognosis. The triple combination array was a useful technique to identify the novel methylated tumor suppressor gene in HCC.

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 323. doi:10.1158/1538-7445.AM2011-323
Abstract 3231: In vitro cross species metabolism of a novel RAF kinase inhibitor phosphate prodrug, ARQ 736
Authors: Hall Terence , Bresciano Karen , Volak Laurie , Wang Yunxia , Zhong Caiyun , Liu Yanbin , Lapierre Jean-Marc , Savage Ronald E. , Chan Thomas C.K.
Published: June 2012

ARQ 736 is a highly-soluble phosphate prodrug that readily undergoes conversion to ARQ 680 in the presence of phosphatases. The active drug, ARQ 680, is a pan-RAF inhibitor that is effective, both in vitro and in vivo, in inhibiting proliferation of cancer cell lines expressing mutant-BRAF (V600E). During preclinical development, the metabolic fate of ARQ 736 and ARQ 680 was evaluated in human, rat, and dog in vitro systems. Based on the data generated, metabolic pathways were proposed for ARQ 736 and ARQ 680. Cross-species metabolic stability studies were conducted in microsomes and hepatocytes. CYP450 reaction phenotyping using 6 recombinant CYP isozymes was conducted to identify the CYP isozyme(s) responsible for the phase one metabolism of ARQ 736 and ARQ 680. ARQ 736 and ARQ 680 were incubated in hepatocytes and liver fractions to evaluate the phase one and phase two metabolites produced in humans rats, and dogs. Additional studies in microsomes and hepatocytes were conducted to evaluate the potential for ARQ 736 or ARQ 680 to inhibit or induce various CYP450 isozymes. Cross-species NADPH-dependent metabolism studies revealed that ARQ 736 and 680 were highly stable in, rat, dog, and human liver microsomes with half-life values >30 min. CYP450 reaction phenotyping indicated that CYP3A4 is the predominant CYP450 isozyme responsible for phase one metabolism of ARQ 736 and 680. Detailed metabolite ID work in hepatocytes identified multiple phase one and two metabolites as a result of oxidations, demethylation and glucuronidation. Metabolic pathways appeared to be similar across all species tested. In conclusion, this research is the first characterization of the metabolism of ARQ 736, a novel pan-RAF inhibitor currently being tested in phase one clinical trial in 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 3231. doi:10.1158/1538-7445.AM2011-3231
Abstract 3239: Shp2 inhibitor activity of estramustine phosphate and its triterpenoid analogues
Authors: Scott Latanya M. , Chen Liwei , Daniel Kenyon G. , Urlam Murali K. , Brooks Wesley H. , Guida Wayne C. , Lawrence Harshani R. , Sebti Said M. , Lawrence Nicholas J. , Wu Jie
Published: June 2012

Shp2 is a non-receptor protein tyrosine phosphatase (PTP) encoded by the PTPN11 gene. Shp2 mediates proliferative signaling induced by growth factors. Gain-of-function PTPN11 mutations that encode constitutively active Shp2 are leukemic oncogenes. In a continuing effort to identify new Shp2 PTP inhibitors, we screened a small molecule library comprising the National Cancer Institute (NCI) Approved Oncology Drug set and the NIH Clinical Collection. After evaluation of initial hits, estramustine phosphate was verified as a Shp2 PTP inhibitor. A focused structure-activity relationship study indicated that the 17-phosphate group is required for the Shp2 PTP inhibitor activity of estramustine phosphate. A search for estramustine phosphate analogs led to identification of two triperpenoids, enoxolone and celastrol, having Shp2 PTP inhibitor activity. With the previously reported PTP1B inhibitor trodusquemine, our study reveals steroids and triterpenoids with negatively charged phosphate, carboxylate, or sulfonate groups as novel pharmacophores of selective PTP inhibitors. These findings point to a rich natural source for discovery of lead compounds of novel PTP inhibitors.

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 3239. doi:10.1158/1538-7445.AM2011-3239
Abstract 325: Mutation analysis of PIK3CA, KRAS, BRAF and Corresponding EGFR status in head and neck squamous cell carcinoma
Authors: Suda Toshihito , Hama Takanori , Okano Susumu , Seino Youichi , Yuza Yuki , Kato Takakuni , Moriyama Hiroshi
Published: June 2012

Background: KRAS, BRAF, PIK3CA activate protein kinase signaling and an important pathway in cancer development. Recent reports show that these downstream signaling molecules are mutated in a number of cancers. However, in head and neck squamous cell carcinoma (HNSCC), the frequency and positions of these molecules mutations are not clear. In this study we detected mutations from the clinical specimens of HNSCC by High-Throughput sequencing KRAS, BRAF, PIK3CA. Next we also intended to discover whether these mutations in downstream signaling molecules of EGFR are corresponding with EGFR status.

Patients and Method: The ninety-six tumors for this study were obtained from HNSCC patients surgically treated at the Department of head and neck surgery, Jikei University School of Medicine, Tokyo. The hot-spot regions in exons 1 and 2 of K-RAS, exon 15 of BRAF, exon 9 and 20 of PIK3CA were amplified by PCR and sequenced directly using DNA from samples. Next, we analyzed expression and phosphorylation of the EGFR protein by Western blotting as well as mutations encoding the hot-spot regions in exons 18 to 21.

Results: We identified mutations in 6 of the 96 patients (6.2%). 3 mutations (G12A) were found in exon 1 of K-RAS. In PIK3CA, 2 mutations were found in exon 9 (E545K) and one was in exon 20(H1047R). No other mutation was found in BRAF. Overexpression of EGFR protein was observed in all clinical samples with these mutations. On the other hand, hyper-phospholyation of EGFR was confirmed in only one sample. Finally, we tried to elucidate associations between these molecular status and patient characteristics and disease-free survival to asses the prognostic values of these molecules factors.

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 325. doi:10.1158/1538-7445.AM2011-325
Abstract 3254: Novel bivalent Smac mimetics as a new class of anticancer agents
Authors: Sun Haiying , Lu Jianfeng , Liu Liu , McEachern Donna , Bai Longchuan , Yang Chao-Yie , Wang Shaomeng
Published: June 2012

Second mitochondria-derived activator of caspase (Smac) is a protein released from mitochondria in response to apoptotic stimuli which functions as an endogenous inhibitor of several members of inhibitor of apoptotic proteins (IAPs), including XIAP, cIAP-1 and cIAP-2. Smac interacts with IAPs by binding of its N-terminal AVPI tetrapeptide to one or more BIR domains in IAPs. We designed and synthesized a series of non-peptidic, cell-permeable, bivalent small-molecules which mimic the dimeric Smac protein for targeting IAPs (bivalent Smac mimetics) and performed extensive evaluations of these bivalent Smac mimetics for their interaction with IAP proteins and their activity and mechanism of function in cancer cells. Our studies show that these Smac mimetics bind to XIAP, cIAP-1 and cIAP-2 with low nano-molar affinities. They function as extremely potent XIAP antagonists by concurrently targeting both the BIR2 and BIR3 domains of XIAP and nullify its inhibition to both caspase-9 and caspase-3/-7 and potently and effectively induce rapid degradation of these two proteins in cancer cells. The most potent bivalent Smac mimetics inhibit cell growth with IC50 values at low nanomolar and effectively induce apoptosis in a subset of cancer cell lines through TNFα dependent mechanism. Furthermore, bivalent Smac mimetics can dramatically enhance the antitumor activity of TRAIL in both TRAIL-sensitive and -resistant cancer cell lines. The combination of one such bivalent Smac mimetic, SM-164 with TRAIL induces rapid tumor regression in vivo in a breast cancer xenograft model in which either agent is ineffective. Mechanistic studies demonstrate that while RIP1 plays a minimal role in TRAIL's activity as a single agent, it is required for the synergistic interaction between TRAIL and SM-164. XIAP and cIAP1 work in concert to attenuate the activity of TRAIL and SM-164 strongly sensitizes TRAIL by concurrently targeting XIAP and cIAP1. Taken together, our data suggest that bivalent Smac mimetics may have a great therapeutic potential for the treatment of human cancer by targeting multiple IAPs, either alone or in combination with TRAIL.

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 3254. doi:10.1158/1538-7445.AM2011-3254
Abstract 3256: New retinoid derivatives as backups of adarotene
Authors: Giannini Giuseppe , Cabri Walter , Brunetti Tiziana , Battistuzzi Gianfranco , Alloatti Domenico , Quattrociocchi Gianandrea , Dallavalle Sabrina , Cincinelli Raffaella , Nannei Raffaella , Vesci Loredana , Pisano Claudio
Published: June 2012

Adarotene (ST1926) (WO03/011808) belongs to a so-called class of atypical retinoids. It represents a new first-in-class potent proapoptotic and cytodifferentiating agent, and was selected by Sigma-Tau for clinical development as a “chemotherapy enhancer” in solid tumors (ovarian ca.).

In pre-clinical models of haematological as well as solid tumors, Adarotene induced a DNA damage response and affected the modulation of cancer cell survival pathways. When used in combination with other anticancer agents, Adarotene showed a significant synergistic effect in most of the combinations and models tested.(1-4) A possible mechanism has also been described.(5-6)

The presence of the phenolic hydroxyl group on Adarotene structure, allowed a rapid O-glucuronidation as a major mechanism of elimination of the drug, favoring a fast excretion of its glucuronide metabolite in the urines.

In order to protect the phenolic hydroxyl and improve the “drug-like” properties of the drug, a series of new derivatives have been synthesized. According to chemical structure, these can be grouped into three classes: ether-, carbamate- and ester-derivatives. All of them have been studied and evaluated for their stability at different pH (1.2 – 6.8 – 7.4). The cytotoxic activity in vitro on NCI-H460 non-small cell lung carcinoma and A2780 ovarian tumor cells was also tested.(7)

A potential back-up of Adarotene has been selected to be evaluated in vivo tumor models.

(1) J. Med. Chem. 2003, 46, 909-912

(2) Blood, 2004, 103, 194-207

(3) J. Med. Chem. 2005, 48, 4931-4946

(4) Bioorganic & Medicinal Chemistry, 2007, 15, 4863-4875

(5) J. Med. Chem. 2008, 51, 5650-5662

(6) J. Comput. Aided Mol. Des. 2010, 24, 943-956

(7) WIPO Patent Application WO2010/072727

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 3256. doi:10.1158/1538-7445.AM2011-3256
Abstract 3259: An efficient total synthesis of Tubulysin B
Authors: Vlahov Iontcho R. , Groaning Michael , Kleindl Paul , Santhapuram Hari Krishna R. , You Fei , Wang Yu , Xu Lecun , Stanford Katheryn , Ritter Allen , Leamon Christopher P.
Published: June 2012

The tubulysins are members of a new class of natural products isolated from myxobacterial species.[1] As cytoskeleton interacting agents, the tubulysins are mitotic poisons that inhibit tubulin polymerization and lead to cell cycle arrest and apoptosis.[2-4] Tubulysins are extremely potent cytotoxic molecules, exceeding the cell growth inhibition of any clinicaly relevant traditional chemotherapeutic e.g. paclitaxel, vinblastine, and epothilones. Furthermore, they are potent against multidrug resistant cell lines.[5]

Structurally, tubulysins are closely related linear tetrapeptides comprised of distinctive, unusual, or hydrophobic amino acid segments: D-N-methyl pipecolinic acid, L-isoleucine, the heterocyclic tubuvalin, and the γ-amino acid tubutyrosine/tubuphenylalanine. The most prominent feature in the molecular architecture of these natural occurring compounds is the acid and base sensitive N-acyloxymethyl substituent (or a N,O-acetal of formaldehyde).

In this poster we present an efficient yet simplified synthesis of Tubulysin B. The optimized synthetic protocol relyes on chemical as well as enzymatic steps and is readily scalable to industrial meaningful quantities. All the synthesized intermediates and final compound are well charectreized by several analytical methods including HPLC, MS, 1H and 13C NMR, etc. Biological activty of synthetic tubulysin B is identical with natural tubulysin B. This synthetic protocol is amenable to all other natural tubulysins and analogs.

[1] F. Sasse, H. Steinmetz, G. Höfle, H. Reichenbach, J. Antibiot. 2000, 53, 879-885.

[2] H. Steinmetz, N. Glasser, E. Herdtweck, F. Sasse, H. Reichenbach, G. Höfle, Angew. Chem. Int. Ed. 2004, 43, 4888-4892.

[3] M. Khalil, F. Sasse, H. Lünsdorf, Y. Elnakady, H. Reichenbach, ChemBioChem. 2006, 7, 678-683.

[4] G. Kaur, M. Hollingshead, S. Holbeck, Schauer-Vikašinovic, R. Camalier, A. Dömling, S. Agarwal, Biochem. J. 2006, 396, 235-242.

[5] For a review see: A. Dömling, W.Richter, Mol. Diversity 2005, 9, 141-147 and references therein.

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 3259. doi:10.1158/1538-7445.AM2011-3259
Abstract 3263: Synthesis of substituted N-(4-(6-methyl-1H-benzo[d]imidazol-2-yl)-5,6-dihydropyridin-1(2H)-yl) benzamide/benzenesulfonamide as anticancer agents
Authors: Farah Said M. , Gangapuram Madhavi , Mateeva Nelly , Mochona Bereket , Ardley Tiffany W. , Redda Kinfe K.
Published: June 2012

There is a strong association between chronic inflammatory conditions in a particular organ and the incidence of cancer specific to that organ. The longer the inflammation persists, the higher is the risk of associated carcinogenesis. Our interest is to synthesis new, potent and safer anticancer agents. The functionalized tetrahydropyridine (THP) ring systems are widely found in biologically active natural products and pharmaceuticals. The pharmacological activities of the THP derivatives depended greatly on the position and nature of the substitutions on the THP ring structure. 2-Substituted benzimidazole derivatives have been found to possess anti-inflammatory, antihistaminic, antimicrobial, anticancer, and cycloxygenase inhibiting activities. It is believed that synthesizing new compounds that contain both the pharmacophores of THP and benzimidazole could have the potential of becoming effective anticancer agents.

6-Methyl-2-(pyridin-4-yl)-1H-benzo[d]imidazole was obtained by the reaction of isonicotinc acid, 4-methyl benzene-1,2-diamine and polyphosphoric acid, which were stirred in an oil bath at 180oC for 2h. Mesitylene sulfonyl chloride was added with stirring to a solution of ethylacetohydroxymate and triethylamine in dimethylformamide at 0o C. Hydrolysis of this compound with the mixture of p-dioxane and 70% perchloric acid for 45 min gave a white solid of MSH. The MSH reacted with 6-methyl-2-(pyridin-4-yl)-1H-benzo[d]imidazole to give 1-amino-4-(6-methyl-1H-benzo[d]- imidazol-2-yl) pyridinium mesitylenesulfonate, which further reacted with different substituted benzoyl chlorides/benzenesulfonyl chlorides in 10% NaOH solution to gave stable benzoyl/benzenesulfonyl imino ylides. These ylides were reduced using sodium borohydride in absolute ethanol and gave the target compounds, substituted N-(4-(6-methyl-1H-benzo[d]imidazol-2-yl)-5,6-dihydropyridin-1(2H)-yl)benzamide/benzenesulfonamides. The cytotoxic effects of these substituted THP derivatives were determined using the MCF-7 estrogen receptor positive breast cancer cells, MDA-MB-231 estrogen receptor negative breast cancer cells, and Ishikawa cells, using the CellTiter-Glo (CTG) luminescent cell viability assay. Two target molecules exhibited strong cytotoxicity activities with significant IC50 values on MCF-7, Ishikawa and MDA-MB-231 cell lines.

This research was supported by the NIH/RCMI Grant G12RR03020 and Pharmaceutical Research Center NIH/NCRR Grant 1 C06 RR12512-01.

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 3263. doi:10.1158/1538-7445.AM2011-3263
Abstract 328: An integrated analysis of three distinct IBC/non-IBC Affymetrix gene expression data sets to study the transcriptional heterogeneity both between IBC and non-IBC and within IBC
Authors: Van Laere Steven J. , Ueno Naoto , Finetti Pascal , Vermeulen Peter B. , Lucci Anthony , Birnbaum Daniel , Robertson Fredika , Iwamoto Takayuki , Van Dam Peter , Woodward Wendy , Viens Patrice , Dirix Luc Y. , Reuben James Reuben , Bertucci François
Published: June 2012

Introduction. Several studies have applied gene expression profiling to inflammatory breast cancer (IBC). Most of these studies were underpowered, mainly because IBC is a rare disease and hence, IBC sample sizes are small. Here, we present an integrated analysis of three distinct gene expression data sets of IBC and non-IBC samples – thus with enhanced power – to further uncover the IBC-specific molecular biology.

Materials & Methods. Three Affymetrix gene expression data sets of 137 IBC and 252 non-IBC samples were integrated. Data were normalized, and genes with high signal-to-noise ratios in at least 1% of the arrays were filtered in. The samples were classified according to their molecular subtypes. IBC-specific heterogeneity was investigated using hierarchical clustering, coupled with silhouette score analysis. Supervised analysis, comparing IBC with non-IBC, was performed in non-stage-matched, stage-matched, and molecular subtype-matched approaches with global testing. IBC-specific activated pathways, miRNA-families, and transcription factors were identified with a target gene analysis approach.

Results. Four robust IBC sample clusters were identified, clearly associated with the molecular subtypes, with a predominance of the combined Basal-like, ErbB2+, and Luminal B subtypes (∼70% vs. ∼40% in non-IBC; P<0.0001). When we compared IBC to non-IBC, stage-matched and non-stage-matched differences were identified (global test, P<0.0001). After comparing IBC with non-IBC samples within each of the molecular subtypes, differences persisted only within the luminal A and normal-like subtypes (global test, P<0.0001 and P=0.046, respectively). Target gene analysis identified two molecular pathways (TGFβ and INFα), 16 transcription factors (e.g. NKX2.2, FOXM1), and 10 miRNA families that were differentially activated in IBC and non-IBC (FDR<0.01) but not in the four identified IBC sample clusters.

Conclusions. This meta-analysis demonstrates that IBC is indeed transcriptionally heterogeneous and that differences between IBC and non-IBC are predominated by the differences in the distribution of the molecular subtypes between IBC and non-IBC. Taking this into account, we were able to more accurately spot IBC-specific changes in pathway, miRNA, and transcription factor activation.

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 328. doi:10.1158/1538-7445.AM2011-328
Abstract 3291: Macrophages and granulocytes promote the malignant glioma phenotype
Authors: de Groot John F. , Liang Jane , Piao Yuji
Published: June 2012

Solid tumors, including glioblastoma, contain large populations of both malignant and non-malignant cells. The tumor microenvironment contains a multitude of different cell types and recent evidence supports the role of CD45+ bone marrow cells such as vascular modulating macrophages. Tumor associated macrophages accumulate in hypoxic or necrotic regions of human tumors where they release VEGF. Recent studies have demonstrated that the number of M2-skewed macrophages (CD163+/CD204+) in human tumors correlates with glioma grade, cell proliferation and microvascular density. Myeloid cells including macrophages and granulocytes are thought to promote the malignant phenotype of many solid tumor types but little is known about their influence on glioma biology.

To evaluate the impact of macrophages and granulocytes on the malignant phenotype, we used co-culture experiments to determine the influence of these cells on glioma cytokine and angiogenic factor release, glioma cell invasion and proliferation, and resistance to chemotherapy. Mouse macrophages or granulocytes (ATCC) were cultured alone or together with U87 or glioma stem cells using a Boyden chamber to maintain cell separation. Conditioned media and RNA were evaluated for expression of both mouse and human chemokine expression using ELISA and quantitative PCR. Co-culture resulted in a significant increase in macrophage VEGF-A, CXCL1, HGF, VEGF-C and PlGF expression and granulocyte IGF1, FGF, CCL11, VEGF-C and VEGF-D compared to baseline suggesting that myeloid cell-glioma crosstalk may promote VEGF-A-independent angiogenesis in glioma tumors. Co-culture promoted glioma cell expression of PDGF, CCL11, CXCL9, and INF-β. We next evaluated the impact of myeloid cells on glioma invasion. At baseline, U87 demonstrates minimal transwell migration in vitro and does not invade normal brain in vivo. When co-cultured, macrophages and granulocytes significantly increased U87 in vitro transwell migration compared to control. Finally, serum free media incubated for 48 hours with mouse macrophages or granulocytes was used as growth medium for U87 cells. Using a SRB cell proliferation assay, conditioned media from granulocytes but not macrophages increased glioma proliferation compared to controls. Conditioned media from both macrophages and neutrophils reduced the sensitivity of glioma cells to temozolomide in vitro.

These results suggest that both macrophages and granulocytes may play an important role in promoting glioma angiogenesis, invasion and resistance to chemotherapy. However, granulocytes were more effective in promoting glioma proliferation. Targeting myeloid cells may be an effective approach to inhibiting the glioma malignant phenotype.

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 3291. doi:10.1158/1538-7445.AM2011-3291
Abstract 3295: Inhibiting vascular morphogenesis in tumors: EGFL7 as a novel therapeutic target
Authors: Yeung Stacey , Smyczek Tanya , Cheng Jason , Lima Anthony , Molina Rafael , Hamilton Patricia , Long Jason , Berry Leanne , Nannini Michelle , Barck Kai , Bou-Reslan Hani , Cao Tim , Ho Calvin , Carano Richard , Kasman Ian , Plowman Greg , Singh Mallika , Ye Weilan , Johnson Leisa
Published: June 2012

EGFL7 is a vascular restricted extracellular matrix protein that is up-regulated during angiogenesis (Campagnolo et al., 2005; Fitch et al., 2004; Parker et al., 2004; Soncin et al., 2003). EGFL7 supports endothelial cell adhesion (Parker et al., 2004; Schmidt et al., 2007) and protects endothelial cells from stress-induced apoptosis (Xu et al., 2008). Inhibition of Egfl7 expression in zebrafish embryos abolished vascular lumen formation and reduced sprouting angiogenesis (Parker et al., 2004).

We developed a panel of anti-EGFL7 monoclonal antibodies that block the adhesive and pro-survival activities of EGFL7. Anti-EGFL7 in combination with anti-VEGF resulted in significant tumor regression in multiple murine xenograft models, whereas anti-VEGF alone only slowed tumor growth in the same models. Anti-EGFL7 monoclonal antibodies also demonstrated prolonged survival and anti-tumor angiogenesis effects in stringent murine genetic tumor models when used as a single agent, and enhancement of anti-VEGF therapy in the combination setting. An ongoing Phase I study is being conducted to evaluate clinical safety, PK, PD and efficacy of anti-EGFL7.

References:

Campagnolo, L., Leahy, A., Chitnis, S., et al. EGFL7 is a chemoattractant for endothelial cells and is up-regulated in angiogenesis and arterial injury. Am J Pathol (2005) 167, 275-284.

Fitch, M. J., Campagnolo, L., Kuhnert, F., et al. Egfl7, a novel epidermal growth factor-domain gene expressed in endothelial cells. Dev Dyn (2004) 230, 316-324.

Parker, L. H., Schmidt, M., Jin, S. W., et al. The endothelial-cell-derived secreted factor Egfl7 regulates vascular tube formation. Nature (2004) 428, 754-758.

Schmidt, M., Paes, K., De Maziere, et al. EGFL7 regulates the collective migration of endothelial cells by restricting their spatial distribution. Development (2007) 134, 2913-2923.

Soncin, F., Mattot, V., Lionneton, F., et al. VE-statin, an endothelial repressor of smooth muscle cell migration. Embo J (2003) 22, 5700-5711.

Xu, D., Perez, R. E., Ekekezie, I., et al. Epidermal growth factor-like domain 7 protects endothelial cells from hyperoxiainduced cell death. Am J Physiol Lung Cell Mol Physiol (2008) 294, L17-L23.

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 3295. doi:10.1158/1538-7445.AM2011-3295
Abstract 330: Gene expression signature characterized by PI3Kinase activation and Stathmin overexpression validates to identify aggressive disease and a potential for PI3Kinase-inhibitors in endometrial carcinomas
Authors: Wik Elisabeth , Trovik Jone , Raeder Maria B. , Mannelqvist Monica , Mauland Karen K. , Oyan Anne M. , Kalland Karl H. , Akslen Lars A. , Salvesen Helga B.
Published: June 2012

Background: We have recently reported a gene expression signature defining an aggressive subgroup of endometrial cancer patients characterized by PI3Kinase activation and Stathmin overexpression, suggesting the potential for PI3Kinase inhibitors against this disease (Salvesen et al, PNAS 2009). In the present study we wanted to validate the value of these measures for PI3Kinase activation prospectively in an independent patient series.

Methods: A prospectively collected, independent patient series of 158 cases of endometrial carcinoma was assessed. Real-time quantitative PCR was performed for the 29 genes in the previously defined gene signature. Stathmin expression in formalin fixed tumor specimens was assessed by immunohistochemistry (IHC).

Results: A high signature score predicted shorter recurrence free (p=0.001) and disease-specific survival (p=0.002). A high score was also significantly associated with high age (p=0.04), high FIGO stage (p=0.03), non-endometrioid histology (p<0.001), high grade (p<0.001) and lymph node metastases (p=0.02). High signature score was also correlated with overexpression of Stathmin in tumor tissue (p≪0.002).

Conclusion: The finding validates that previously identified markers for PI3Kinase activation identify aggressive phenotype in endometrial cancer. This further supports a potential for PI3Kinase-inhibitors in the treatment of metastatic endometrial carcinoma. We suggest that clinical trials with drugs targeting the PI3Kinase pathway involve stratification related to biomarkers, including the expression levels for this gene signature and Stathmin.

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 330. doi:10.1158/1538-7445.AM2011-330
Abstract 3305: Trim3, the human homolog of Drosophila brat, regulates asymmetric division in glioblastoma stem cells
Authors: Chen Gang , Rong Y , Rahman F , Tucker-Burden C , Hadjipanayis C , Van Meir E G. , Brat D J.
Published: June 2012

Glioblastoma (GBM) is the most common and malignant primary brain tumor. The recently described stem cell component of malignant gliomas is biologically critical, yet mechanisms that underlie its regulation are not understood. Mutations in Drosophila Brain tumor (brat) result in a massively enlarged fly brain that consists of a neuroblast cell population with neoplastic growth properties that has lost the ability to divide asymmetrically. Brat has been shown to promote asymmetric cell division and direct neural differentiation at least partially through its translational suppression of Myc. Here we investigated the role of brat homologs for their regulation of glioma behavior, especially as related to asymmetric division of glioma stem cells. We found that the human brat homolog, Trim3, is highly expressed in normal brain and that a substantial subset of GBMs (25-30%) show loss of heterozygosity (LOH) of chromosome 11p15.5, the site of Trim3. Trim3 expression was markedly reduced in human GBM cell lines compared to human astrocytes and reduced in human GBMs specimens compared to normal brain, consistent with results from mining existing gene expression data sets (oncomine). Since Drosophila brat is critical to neural differentiation and regulates Myc, we explored whether human Trim3 might regulate glioma stem cell properties by its regulation of Myc. We found that the forced expression of Trim3 in human GBM cell lines led to reduced Myc protein expression. This inverse relationship between Trim3 and Myc also held true in GBM samples. We used a lentiviral system to stably express Trim3 in GBM cell lines and neurospheres and found that Trim3 expression reduced glioma proliferation and colony formation in vitro and also led to smaller and less frequent neuropheres. In vivo, Trim3 suppressed tumor growth in an orthotopic xenograft model and led to prolonged survival. Trim3 expression was associated with reduced expression of nestin and CD133 by GBM neurosphere cultures, suggesting that its loss may enrich the stem cell compartment. Moreover, Trim3 expression by neurosphere stem cells favored a pattern of asymmetric cell division in GBM. Thus, Trim3 appears to direct asymmetric cell division in glioma stem cells and its loss is associated with stem cell enrichment and greater biologic potential.

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 3305. doi:10.1158/1538-7445.AM2011-3305
Abstract 3308: Endothelin 3/endothelin receptor B signaling pathway blockade depletes radio-chemoresistant glioblastoma stem cells
Authors: Ye Fei , Yamada Kazunari , Liu Yue , Choe Jinny , Dembo Anna , Tso Jonathan L. , Cloughesy Timothy F. , Schaue Dörthe , McBride William , Liau Linda M. , Tso Cho-Lea
Published: June 2012

Background: Patients with glioblastoma multiforme (GBM) despite being treated with gross total resection and post-operative temozolomide (TMZ) plus radiation therapy (RT), will almost always develop tumor recurrence.

Hypothesis and objective: We hypothesize GBM stem cells (GSC) are responsible for post-treatment tumor recurrence and blockade of endothelin-3 (EDN3)/endothelin receptor B(EDNRB) signaling, a signaling pathway for neural crest development that hyperactivated in GSC, will enhance TMZ/RT treatment efficacy for GSC.

Materials and Methods: Patient tumor-derived GSC were seeded at clonal density and subjected to radiochemotherapy at dose and schedule comparable to patient treatment. Treatment efficacy and synergy of EDNRB antagonist (BQ788) plus TMZ/RT were evaluated by the clonogenic efficiency, cell proliferation, and cell apoptosis of GSCs.

Results: GSC are resistant to TMZ treatment alone (10μM), while treatment with BQ788 (75μM) alone can restrict cell migration, decrease clonogenic efficiency, and induce cell apoptosis. The RT displays the highest treatment efficacy on GSC when compared to TMZ or BQ788 alone and addition of TMZ to RT is slightly synergistic. Although only few small tumor spheres were detected when treated with RT or RT/TMZ, the survival clones regain proliferative activity and possess enhanced clonogenicity and anti-apoptotic ability when compared to untreated GSC. Treatment efficacy was greatly improved when GSC treated with BQ788 combined with RT/TMZ and no cells with self- renewal capacity were detected or recovered from the treatment.

Conclusion: Subsets of GSC clones are resistance to standard RT/TMZ treatment and possess enhanced tumor sphere-forming capacity. Incorporate EDN3/EDNRB antagonist to a standard radiochemotherapy provide a significant treatment synergy in depletion of functional GSC and thus highlights a novel therapeutic strategy for the prevention of GSC-mediated tumor occurrence and recurrence.

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 3308. doi:10.1158/1538-7445.AM2011-3308
Abstract 3311: IL6-Stat3 signaling promotes glioma stem cell (GSC) survival under hypoxia environment through inhibition of cell-death inducing gene Bnip3
Authors: Wang Hui , Wang Xiao-Fan , Rich Jeremy , Wang Qinhong , Hjelmeland Anita
Published: June 2012

Glioblastomas (GBMs) are the most common and lethal brain tumor in adults. In rapid-growing tumors like malignant glioma, hypoxia is an unavoidable condition even with aggressive angiogenesis. Recent studies implicate an important role for a restricted population of neoplastic cells (glioma stem cells, GSCs) in glioma maintenance and recurrence. However, how these glioma stem cells survive and escape from harsh conditions like hypoxia is barely known. Our previous study and others’ have shown that GSCs preferentially express the interleukin 6 (IL6) receptors and Stat3 signaling is preferentially activated in GSCs. We further found that perturbation of IL6 signaling in GSCs reduced Stat3 activation, and small molecule inhibitors of Stat3 potently induced GSC cell death partially through induction of a BCL2 family member Bnip3. Despite its controversial role in cell survival, massive expression of Bnip3 is a well-studied mediator of cell death in hypoxic tumor cells. Furthermore, we found that enhanced IL6-Stat3 signaling in GSCs could counter the induction of Bnip3 under hypoxia, which provided an extra survival advantage for these glioma stem cells under severe hypoxic condition. These data indicated that Stat3 is a downstream mediator of pro-survival IL6 signals in GSCs partially through suppressing Bnip3 expression, especially under hypoxia condition. Together, our data indicate that IL6-Stat3 signaling contributes to glioma malignancy through the promotion of GSC survival under harsh conditions like hypoxia, and that targeting IL6-Stat3 signaling pathway may offer benefit for glioma 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 3311. doi:10.1158/1538-7445.AM2011-3311
Abstract 3322: Downregulation of XIAP by cord blood stem cells is associated with inhibition of heat shock protein 90 in glioblastoma
Authors: Dasari Venkata Ramesh , Velpula Kiran Kumar , Dinh Dzung H. , Tsung Andrew J. , Rao Jasti S.
Published: June 2012

Heat shock protein 90 (HSP90) is highly expressed under stressful conditions in eukaryotic cells showing chaperone activity including the suppression of protein aggregation and signaling pathways. HSP90 is upregulated in various human tumors, including glioblastoma, and targeting its function might provide new therapeutic opportunities. Previously, we have shown that human umbilical cord blood-derived mesenchymal stem cells (hUCBSC) downregulate XIAP in glioblastoma cells in vitro and in vivo. In the present study, we report that downregulation of XIAP by hUCBSC is associated with inhibition of HSP90. Under in vivo conditions, hUCBSC induce apoptosis by downregulation of XIAP in which XIAP migrates from the cytosol to the nucleus in mice brain xenografts. Immunoprecipitation experiments proved that SMAC is associated with Cytochrome C and XIAP in the mitochondria, indicating mitochondrial apoptosis. Simultaneously, cytosolic HSP90 is downregulated and co-localized with TNFR2 in hUCBSC-treated tumors, proving that TRAIL-mediated apoptosis is taking place in hUCBSC-treated tumor xenografts. Also, expression of mitochondrial HSP90 (TRAP-1) is downregulated in glioma cells. The downregulation of XIAP by hUCBSC is shown to be under the transcriptional control of SP1 under both in vitro and in vivo conditions. These results have been confirmed by using shRNA for XIAP. Taken together, these results prove that hUCBSC are efficient in the inhibition of HSP90, and hUCBSC have the potential to be therapeutic tools for the treatment of glioblastoma.

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 3322. doi:10.1158/1538-7445.AM2011-3322
Abstract 3327: Leptin receptors are expressed in breast cancer stem cells and leptin promotes their self-renewal and survival in mice
Authors: Zheng Qiao , Smith Sarah , Zhu Jinling , Downs-Kelly Erin , Hursting Stephen D , Rich Jeremy N , Berger Nathan A , Reizes Ofer
Published: June 2012

Cancer stem cells (CSCs) are highly important in Breast cancer because they represent a subset of cancer cells that are generally undifferentiated, capable of self-renewal, resistant to therapy, and contribute to post-therapeutic tumor regrowth. CSCs can drive the malignant process and may generate the non-renewing differentiated cancer cells that comprise the bulk of the tumor. Here we show that in the Murine Mammary Tumor Virus (MMTV)-Wnt-1 transgenic mouse, a model of basal-like human breast tumors, leptin promotes tumorigenesis and the survival of breast CSCs. We show that leptin deficiency results in reduced outgrowth and burden of transplanted tumors. Second, leptin deficiency leads to depletion of cancer cells with a stem cell molecular signature. Third, the functional importance of the reduced cells is demonstrated by the fact that tumors from leptin-deficient mice led to reduced tumor outgrowth in vivo. Fourth, breast CSC enriched populations express the full-length leptin receptor. Finally, leptin promotes the viability of CSC enriched populations and increases tumorsphere generation in vitro. These results identify a critical role for leptin stimulation of breast CSCs and provide a strong suggestion that interfering with leptin signaling either via receptor blockade or a downstream pathway may provide therapeutic benefit.

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 3327. doi:10.1158/1538-7445.AM2011-3327
Abstract 3330: MicroRNA100 regulates breast cancer stem cells
Authors: Liu Suling , Bai Shoumin , Ke Jia , Martin-Trvino Rachel , Patel Shivani H. , McDermott Sean , Luther Tahra , Heath Amber , Ibarra Ingrid , Korkaya Hasan , Clouthier Shawn G. , Hannon Gregory , Wicha Max S.
Published: June 2012

There is increasing evidence that many human cancers, including breast cancer, are driven and maintained by a cellular subcomponent that displays stem cell properties. These “cancer stem cells” (CSCs) may also mediate tumor metastasis and contribute to treatment resistance and relapse. Our laboratory has identified cellular markers and developed in vitro and mouse models to isolate and characterize normal and malignant human mammary stem cells. We have previously demonstrated that breast cancer cell lines contain subpopulations with stem cell properties that can be isolated by virtue of their expression of Aldehyde dehydrogenase (ALDH) as assessed by the Aldefluor assay. We demonstrated, among microRNAs differently expressed in Aldefluor-positive and Aldefluor-negative populations of breast cancer cell lines we identified micrRNA100 (mir100). We found that mir100 is significantly increased in Aldefluor-negative compared to Aldefluor-positive populations. Utilizing a tetracycline inducible lentivirus driving mir100 expression, we found that induction of mir100 expression decreased the ALDH-positive population of SUM159 cells in vitro as well as in mouse xenografts where this reduction was associated with decreased tumor growth. Furthermore, induction of mir100 expression immediately upon orthotopic implantation or intracardiac injection completely blocked subsequent tumor growth and metastasis formation. These studies demonstrate that mir100 plays a functional role in the self-renewal and differentiation of breast cancer stem cells. Furthermore, the TET-inducible mir100 system allows for controlled regulation of the cancer stem cell population providing a valuable model to simulate the effects of CSC-directed therapies on breast cancer growth 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 3330. doi:10.1158/1538-7445.AM2011-3330
Abstract 3334: Repression of mammary stem/progenitor cells by p53 is mediated by notch and separable from apoptotic activity
Authors: Tao Luwei , Roberts Amy L. , Dunphy Karen A. , Bigelow Carol , Yan Haoheng , Jerry D. Joseph
Published: June 2012

Breast cancer is the most common tumor among women with inherited mutations in the p53 gene (Li-Fraumeni syndrome). The tumors represent the basal-like subtype which has been suggested to originate from mammary stem/progenitor cells. In mouse mammary epithelium, mammosphere-forming potential was increased with decreased dosage of the gene encoding the p53 tumor suppressor protein (Trp53). Limiting dilution transplantation also showed a 3.3-fold increase in the frequency of long-term regenerative mammary stem cells in Trp53-/- mice. The repression of mammospheres by p53 was apparent despite the absence of apoptotic responses to radiation indicating a dissociation of these two activities of p53. The frequency of long-term label-retaining epithelial cells (LRECs) was decreased in Trp53-/- mammary glands indicating that asymmetric segregation of DNA is diminished and contributes to the expansion of the mammary stem cells. Treatment with an inhibitor of γ-secretase (DAPT) reduced the number of Trp53-/- mammospheres to the level found in Trp53+/+ cells. These results demonstrate that basal levels of p53 restrict mammary stem/progenitor cells through Notch and that the Notch pathway is a therapeutic target to prevent expansion of this vulnerable pool of cells.

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 3334. doi:10.1158/1538-7445.AM2011-3334