bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3063: Mcl-1 dependency is a predictive biomarker for apoptotic induction by short-term dinaciclib (SCH 727965) treatment
Authors: Booher Robert , Hirsch Heather , Strack Peter , Zawel Leigh , Fawell Stephen
Published: August 2012

Dinaciclib (SCH 727965) is a potent and selective inhibitor of the cyclin-dependent kinases (CDKs) 1, 2, 5 and 9 undergoing clinical testing against a range of solid and hematologic malignancies. From preclinical studies, more than 140 cell lines have been profiled for dinaciclib response in long-term (>72 hrs) viability or clonogenicity assays with >97% of the lines showing IC50 β25 nM. This uniformly low nM potency is likely due to repression of both cell cycle progression and transcription through inhibition of CDK1/2 and CDK9, respectively. CDK9 phosphorylation of the RNA pol II (RNAPII) at Ser2 and 5 is required for transcriptional initiation and elongation. We and others have observed rapid CDK9-dependent effects on cells after short-term dinaciclib exposure, including loss of RNAPII Ser2 phosphorylation followed by rapid elimination of the short half-life, pro-survival protein Mcl-1. Since a cancer cell's ability to avoid apoptosis is dependent on the balance of several Bcl-2 antiapoptotic family members, including Bcl-2, Bcl-xL and Mcl-1, we hypothesized that Mcl-1 dependent cell lines would be more sensitive to dinaciclib treatment. Moreover, we anticipated that this differential sensitivity could be discriminated from longer-term inhibitory cell cycle effects by conducting short-term dinaciclib exposure assays. Here we report the activity of dinaciclib to induce apoptosis on a panel of 25 human solid tumor cell lines with varying levels of Mcl-1 dependency. Mcl-1 dependency in solid tumor cell lines has been reported to correlate with the Mcl-1 to Bcl-xL mRNA ratio or the level of Mcl-1 gene amplification. Cell viability was assessed after an 18 hour, 100 nM dinaciclib treatment while target engagement and induction of apoptosis was determined after 8 hours. With one exception, all cell lines showed potent CDK9 target engagement as determined by loss of RNAPII Ser2 phosphorylation and corresponding reduced Mcl-1 protein levels. We observed that loss of cell viability, measured by ATP content, directly correlated with the Mcl-1/Bcl-xL mRNA ratio. A dramatic increase in PARP cleavage was also observed in cell lines with the highest Mcl-1/Bcl-xL mRNA ratio. Furthermore, the extent of PARP cleavage correlated with levels of caspase-3/7 activity. Bcl-2 levels did not significantly impact the dinaciclib response. These data provide a rationale for utilizing Mcl-1 dependency as a predictive biomarker for dinaciclib anti-cancer response in solid tumor malignancies.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3063. doi:1538-7445.AM2012-3063
Abstract 3116: Patterns of genomic loss of heterozygosity predict homologous recombination repair defects in ovarian cancer
Authors: Timms Kirsten M. , Abkevich Victor , Hennessy Bryan T. , Potter Jennifer , Carey Mark S. , Meyer Larissa A. , Broaddus Russell , Lu Karen H. , Jammulapati Srikanth , FitzGerald Lisa , Krivak Thomas C. , DeLoia Julie A. , Gutin Alexander , Mills Gordon B. , Lanchbury Jerry
Published: August 2012

Introduction: BRCA1 and BRCA2 are key members of the homologous recombination (HR) pathway. Mutations in these genes and other HR pathway defects have potential therapeutic relevance when used to support agents that introduce or exploit double-strand DNA breaks. This study examines the association between HR deficiency and genomic patterns of loss of heterozygosity (LOH). Methods: Ovarian tumors from two independent datasets were characterized for germline and somatic defects in BRCA1 and BRCA2. Whole genome LOH profiles were generated using Affymetrix SNP arrays. Publically available data was downloaded from the TCGA website for a third independent ovarian cancer dataset. RAD51C promoter methylation was assayed in two of the datasets. Comprehensive profiling of BRCA1 and BRCA2 defects, and genome wide LOH was also performed on approximately 70 breast, ovarian, colon and pancreatic cell lines. Results: Examination of the pattern of LOH within ovarian tumors with BRCA1, BRCA2, or RAD51C defects compared to tumors without defects in these genes has resulted in the development of a homologous recombination deficiency (HRD) score that has highly significant association with HRD (p=9*10-11). An intermediate class of LOH sizes (>15 Mb but less than a whole chromosome) is highly positive correlated with defective HR, suggesting this class of LOH exists due to double strand DNA break formation and requires repair by HR. The HRD score was validated in two independent ovarian cancer datasets (p=2*10-7 and 9*10-29), and successfully identified breast and pancreatic cell lines with BRCA defects, suggesting it will be effective across multiple tumor types. Conclusions: BRCA1 or BRCA2 mutation carriers have improved outcomes following treatment with DNA damaging agents such as platinum salts, and preclinical studies have demonstrated PARP inhibitor efficacy in BRCA1 or BRCA2 deficient cells. HR deficiency in ovarian cancer is not solely due to germline BRCA1 and BRCA2 mutations, and HR deficiency is not unique to ovarian tumors. Each type of cancer is likely to have a unique spectrum of genetic variants resulting in HRD. The HRD score appears capable of detecting HRD regardless of etiology or mechanism. This score could have clinical utility in breast and ovarian cancer, and could be used to target the use of PARP inhibitors and platinum salts in other cancers.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3116. doi:1538-7445.AM2012-3116
Abstract 312: Blockade of tumor-infiltrating myeloid cells by inhibiting CSF-1 receptor overcomes tumor regrowth after radiotherapy
Authors: Xu Jingying , Escamilla Jemima , Wu Lily
Published: August 2012

Radiotherapy is one of the major frontline treatments for prostate cancer (PCa). However, a large proportion of patients suffer from tumor recurrence after radiotherapy. Tumor-infiltrating myeloid cells (TIMs), including CD11b+F4/80+ tumor-associated macrophages (TAM) and CD11b+Gr-1+ myeloid-derived suppressive cells (MDSCs), play critical roles in promoting tumoral angiogenesis, tissue remodeling and immunosuppression. Recently several reports suggested that TIMs might play a critical role in mediating tumor regrowth after radiation therapy. Here, we showed that there is enhanced recruitment of both TAMs and MDSCs after 5 days of 3 Gy radiation treatment in subcutaneously implanted RM-1 syngeneic PCa model. Infiltration of two MDSC subsets, CD11b+Gr-1loLy6Chi mononuclear-MDSC (MO-MDSC) and CD11b+Gr-1hiLy6Clo polymorphnuclear-MDSC (PMN-MDSC), are both increased. Although treatment is localized to the tumor site, the effects of radiation are systematic. Dramatic increases of MDSCs and TAMs were also observed in the spleen, lymph nodes and peripheral blood. To better understand the mechanism of enhanced recruitment of myeloid cells to tumors, we performed RT-PCR analysis on tumor tissues for commonly known factors that mediate the recruitment of myeloid cells. Indeed, we found that macrophage colony stimulating factor 1 (M-CSF-1 or CSF-1) increases by 2 fold in irradiated tumors compared to non-irradiated tumors. In vitro migration assays using conditioned medium from irradiated tumor cells increases migration of RAW246.7 macrophages by 4 fold, and this increase is completely blocked by the very selective CSF1R inhibitor, GW2580. Immunohistochemical analysis confirmed the induction of TAMs and MDSC recruitment to tumors, and the effectiveness of GW2580 in blocking their infiltration. Most importantly, combination therapy using GW2580 with radiation synergistically suppressed tumor growth and recurrence. This data highlights the importance of CSF1/CSF1R signaling in the recruitment of TIMs post-radiation treatment and their significant role in promoting tumor re-growth. Moreover, our data could potentially provide a more effective and durable treatment strategy for PCa patients.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 312. doi:1538-7445.AM2012-312
Abstract 3120: Functional characterization of ERCC1 isoforms in NSCLC
Authors: Friboulet Luc , Thomale Juergen , Olaussen Ken , Ponsonnailles Florence , Dorvault Nicolas , Commo Frederic , Saulnier Patrick , Vagner Stephan , NAKOUZI Nader , André Fabrice , Soria Jean-Charles
Published: August 2012

Background: ERCC1 is a DNA repair protein whose expression is a prognostic and predictive biomarker of chemotherapy effect in NSCLC. Five alternative transcripts of the ERCC1 gene have been described or hypothesized to exist (isoforms 201 to 205). Nevertheless, little is known about the function and relative expression of these isoforms. We explored 3 main questions: a- Which ERCC1 isoforms are detected by clinically validated antibodies? b- Which isoforms are expressed in lung cancer cell lines and patient samples? c- Which isoforms are functional for removal of cisplatin DNA-adducts and potentially relevant as predictors of cisplatin resistance? Methods: To determine the specificity of anti-ERCC1 antibodies, a screening of isoform specific peptide sequences was achieved using a peptide array (Epitope Mapping CelluSpots™). The expression of ERCC1 isoforms was evaluated in 20 lung cancer cell lines and in 123 NSCLC patients by qRT-PCR. We established a functional DNA repair assay by monitoring the accumulation of cisplatin adducts over time with a specific antibody RC-18 (courtesy of Juergen Thomale, Germany). To investigate isoforms functionality, we created stable ERCC1 attenuated A549 cells with Zinc Finger Nucleases technology (Sigma). Using lentiviral constructs we rescued ERCC1 isoforms expression. Results: The 2 monoclonal antibodies, clone 8F1 and 3H11 recognize a conserved region shared by all isoforms of ERCC1 protein, and so, are unable to distinguish the different isoforms. The 5 isoforms were all detected at mRNA level, both in cancer cell lines and in patient tissues. Interestingly, a significant increase in isoform 201 expression was observed in all tumor tissues compared to normal counterparts. We obtained 7 A549 clones harboring a 85-95% decrease in ERCC1 expression. In these cells we observed a 7-fold increase in IC50 of cisplatin accompanied by a significant decrease in the rate of nucleotide excision repair (NER). We are currently exploring isoform function by analyzing DNA repair efficiency in the different stable cell lines. Conclusions: Alternative roles for ERCC1 beyond NER are currently emerging. Our 7 A549 clones, with stable and strong ERCC1 attenuation, provide a very promising tool to analyse all functions of ERCC1. Since most antibodies used to test ERCC1 positivity in patients do not distinguish the different isoforms, it is urgent to characterize their function further, particularly the role of isoform 201 overexpression in NSCLC tumors.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3120. doi:1538-7445.AM2012-3120
Abstract 3149: MicroRNA-141 inhibits proliferation and invasion by suppressing the Wnt signaling pathway in renal cell carcinoma
Authors: Yamamura Soichiro , Sharanjot Saini , Shahana Majid , Hirata Hiroshi , Ueno Koji , Chang Inik , Chiyomaru Takeshi , Tanaka Yuichiro , Dahiya Rajvir
Published: August 2012

MicroRNAs (miRNAs) are highly conserved, single stranded, non-coding RNAs of approximately 22 nucleotides that regulate gene expression by posttranscriptional silencing of specific target mRNAs, by repressing translation or cleaving RNA transcripts. miRNAs regulate diverse cellular processes such as cell-cycle progression, proliferation, apoptosis and development. miRNAs have been shown to function as oncogenes or tumor suppressor genes. MicroRNA-141 has recently been reported to play a role in the epithelial to mesenchymal transition (EMT) process and the expression of miR-141 is inversely correlated with tumorigenicity and invasiveness in several human cancers. The function of miR-141 in renal cell carcinoma has not been studied, therefore we studied the functional effects of miR-141 on renal cell carcinoma. We found that miR-141 was down regulated in human kidney cancer tissues compared with adjacent normal tissues. We also found that miR-141 was down regulated in renal cell carcinoma human cell lines, 769P, 786-O and ACHN compared with normal kidney cell line, HK-2. Transfection of miR-141 into renal cell carcinoma cells promoted apoptosis, inhibited in vitro cell proliferation and inhibited cell invasion. We found that miR-141 has a binding site in 3′UTR of the TCF7L2 (Transcription factor 7-like 2) gene which can activate the canonical Wnt pathway. miR-141 was found to down regulate expression of TCF7L2 by targeting its 3′ UTR as shown by luciferase reporter assays in renal cell carcinoma. Luciferase assays using T cell factor (TCF) reporter plasmids also showed that miR-141 represses TCF/LEF transcriptional activity, indicating that miR-141 suppresses the canonical Wnt pathway in renal cell carcinoma. Our results demonstrate that miR-141 suppresses malignancy by targeting TCF7L2 and inhibiting the Wnt signaling pathway in renal cell carcinoma. Source of Funding: Grants R01CA138642, VA Research Enhancement Award Program (REAP), and VA Merit Review grants

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3149. doi:1538-7445.AM2012-3149
Abstract 3150: miR-100 in ovarian cancer cell lines
Authors: Howell Viive M. , Dickson Kristie-Ann , Kan Casina W. S. , Hahn Michael A. , Marsh Deborah J.
Published: August 2012

MicroRNA (miRNA), small non-coding RNA sequences that regulate gene expression, have the potential to function as tumor suppressors or oncogenes. To determine the contribution of miRNA to ovarian oncogenesis this project sought to identify dysregulated miRNA and their targets in ovarian cancer cell lines. miRNA expression profiling was performed on 6 serous epithelial ovarian cancer (SEOC) cell lines and normal ovarian surface epithelial (OSE) cells. miRNAs differentially expressed between SEOC and OSEs were selected for further analyses and candidate target genes identified by prediction algorithms. miRNA expression was restored by transient transfection of miRNA mimics or stable transduction of lentiviral vectors expressing miRNA precursors. The effects on the expression of 2 predicted targets, SMARCA5 and FRAP1, were investigated using a luciferase reporter system, quantitative RT-PCR (qPCR) and immunoblotting. The effect of miRs on cellular proliferation was also explored. Expression profiling identified miR-100 as having >5-fold lower expression in SEOC vs OSE cells. Seed sequences or binding sites for miR-100 were identified in the 3′UTRs of SMARCA5 and FRAP1. Using a luciferase expression system in OVCAR-3 cells, a miR-100 mimic repressed expression of SMARCA5 3′UTR by 40 % (p<0.0001), and FRAP1 3′UTR by 30% (p<0.0005). No effect of the mimic was observed when the predicted miR-100 binding sites in each gene's 3′UTR were mutated. Stable transduction of OV202 cells with pre-miR-100 resulted in a 3-5 fold increase in miR-100 expression. This stably transduced SEOC cell line showed decreased cellular proliferation, and repressed transcript and protein levels of SMARCA5/SNF2H and FRAP1/mTOR compared with the cell line transduced with the empty lentiviral vector. We identified miR-100 as a modulator of cellular proliferation in OV202 cells, possibly through regulation of SNF2H, a chromatin remodelling factor, and mTOR a phosphatidylinositol kinase-related kinase. miR-100 is located in an area of frequent chromosomal loss in aggressive SEOC suggesting a mechanism for reduced expression in these cells. Future work will investigate the potential of miR-100 as a novel therapeutic target for the treatment of ovarian cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3150. doi:1538-7445.AM2012-3150
Abstract 3159: Differential miRNA expression profiling of rectal and colon cancers using deep sequencing
Authors: Chen Zhenbin , Mu Bing , Qin Hanjun , Wang Jinhui , Wu Xiwei , Gao Harry , li Wenyan , Garcia-Aguilar Julio
Published: August 2012

Background: miRNAs are small regulatory elements involved in regulating gene expression. They have been shown to play a role in tumor initiation, progression, and metastasis in many cancers and their expression is often tissue-specific. Cancers of the large bowel are often grouped together as colorectal cancers; but tumors from different locations require different treatment and have different prognosis and molecular characteristics. We tested the hypothesis that cancers of the colon and rectum have different molecular miRNA profiles that can be used to distinguish between these cancer types. Methods: We collected 40 pretreatment rectal cancer biopsies and 20 pretreatment surgical colon cancer specimens. All samples were formalin fixed and paraffin embedded (FFPE). Tumor cells were microdissected manually under inverted microscopy and total RNA was extracted. A miRNA expression profile for rectal and colon cancer was generated using deep sequencing, and miRNA expression in each cancer type was compared using T-test and multiple testing (Q-bound <0.05) to determine significant miRNA expression changes in rectal versus colon cancer. Results: 143 miRNAs were differentially expressed between rectal and colon cancer tissues (Q-bound <0.05); 106 miRNAs were up-regulated and 37 were down-regulated in rectal cancer compared to colon cancer. Thirteen miRNAs showed a greater than 2-fold increase in expression in rectal cancer compared to colon cancer (mir-1291, mir-1248, mir1973, mir-3653, mir-483-5p, mir-720, mir-3647-3p, mir3687, mir-3607-3p, mir4286, mir-1274b, mir-483-3p, and mir3648). Six miRNAs showed a greater than 1.5-fold decrease in rectal cancer compared to colon cancer (mir-374a, mir-3613-5p, mir-362-3p, mir-340, mir-3, and mir-424). Based on these significant expression differences, the unique miRNA expression profiles of rectal and colon cancer were used to separate these cancers by hierarchical cluster analysis (p<0.0001). Conclusions: We identified a number miRNAs that are differentially expressed between rectal and colon cancer. These tissue-specific miRNA expression profiles may help us understand the biological differences between colon and rectal cancer, which may lead to improved treatments.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3159. doi:1538-7445.AM2012-3159
Abstract 3209: Metformin suppresses the growth and modulates the metabolic profiles of pancreatic cancer cells under hypoxia in an IKKα-dependent manner
Authors: Emami-Shahri Nia , Maniati Eleni , Bossard Maud , Hagemann Thorsten
Published: August 2012

Nuclear factor-βB; (NF-κB) controls a range of genes involved in inflammation, cell survival, growth and apoptosis. The dysregulated NF-κB pathway has been found to be involved in carcinogenesis and cancer progression. An IKKα-deleted mouse pancreatic cancer cell line was generated using a lentiviral construct with an shRNA. We investigated the effects of metformin, an anti-diabetic drug, on the cell proliferation and metabolism of these cells, under normoxic and hypoxic conditions. A high dose (5 mM) of metformin under normoxic conditions suppressed the growth of both wild-type and IKKα knockdown cell types; however, under hypoxic conditions IKKα knockdown cells were more sensitive to treatment than their wild-type counterpart (p < 0.001). Metabolic assays revealed that metformin treatment under normoxic conditions decreased the oxidative phosphorylation (OXPHOS) whilst increasing the lactate production of both cell types to similar levels. Under hypoxia, metformin treatment of both cancer cells resulted in decreased OXPHOS, with a more profound effect on the IKKα knockdown cells, and a significant decrease in lactate production in the IKKα knockdown cells only (p < 0.001). We believe our findings have implications for understanding the molecular mechanisms of metformin as a therapeutic agent in cancer treatment.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3209. doi:1538-7445.AM2012-3209
Abstract 3216: Cytosolic leucine metabolism regulates the expression of prometastatic proteins: The genesis of a new druggable target
Authors: Papathanassiu Adonia E. , Vu Hong A.
Published: August 2012

Branched chain amino acid metabolism begins with the reversible transamination of the amino acids to the corresponding α-keto acids. The reaction is catalyzed by the enzyme Bcat. The cytosolic form of this enzyme, also known as Bcat1, is thought to be expressed only in embryonic tissues, in select adult organs (brain, ovary), and in c-myc induced brain tumors. In the current study, Bcat1 is shown to be expressed by a variety of tumor cell lines including MCF-7, MDA-MB-231, MDA-MB-435, and HT-29. Its expression appears to be associated with the ability of the cells to uptake glucose since treatment of MDA-MB-231 with 2-deoxyglucose leads to the suppression of the cellular Bcat1 levels in vitro and in vivo. In the cytosol, Bcat1 appears to complex with CD147, a protein known to promote cancer growth and metastasis through multiple pathways. Silencing of Bcat1 in MDA-MB-231 and MDA-MB-435 cells results in the downregulation of CD147, while inhibition of Bcat1 by ERG-240, a small molecule structurally related to leucine, leads to downregulation of the cell surface expressed CD147 and its transmembrane partner monocarboxylate transporter-4 (MCT-4) in MDA-MB-231 breast cancer cells. MCT-4 is known to mediate the expedient removal of lactate, a cytotoxic by-product of cancer glycolysis, from the cytosol. Furthermore, treatment of MDA-MB-435 cells with 10 mM ERG-240 results in downregulation of matrix metalloproteinase 9 (MMP-9) production, while treatment of normal adult fibroblasts with either ERG-240 or conditioned media of MDA-MB-435 cells previously exposed to ERG-240 results in downregulation of MMP-2 production. The later is attributed to the ability of ERG240 to suppress the presence of soluble CD147 in the conditioned media. Exocytosis of CD147 has been implicated in the formation of the pre-metastatic niche through inductions of MMPs and VEGF. Inhibition of Bcat1 by ERG-240 suppresses cell proliferation in various tumor cell lines with an IC50 value of 5 to 10 mM. ERG240-induced inhibition of MDA-MB-231 cell migration is also observed. The results from an on-going in vivo study are presented. In summary, our experiments suggest that: a) metabolism of branched chain amino acids is intimately linked with the expression of proteins controlling metastatic activities of tumor cells and b) Bcat1, which is not expressed by normal adult tissues, is an attractive target for the development of new, non-toxic anticancer therapies.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3216. doi:1538-7445.AM2012-3216
Abstract 3223: Magnetic resonance spectroscopic imaging of lactate in LDH-A silenced metastatic breast tumors
Authors: Rizwan Asif , Serganova Inna , Ni Xiaohui , Thakur Sunitha , Zakian Kristen L. , Blasberg Ronald , Koutcher Jason
Published: August 2012

Glucose and glutamine are the major carbon sources for rapidly proliferating tumors. They provide the precursors for proteins, lipids, and nucleic acids, and as well as metabolic-reducing capability (NADPH). Pyruvate is mainly derived from glucose and glutamine metabolism and can be converted to lactate by the lactate dehydrogenase (LDH) complex or enter the TCA cycle for conversion to CO2 and ATP. Recently, a link between tumor lactate levels (monitored by MRSI), LDH-A expression and tumor phenotype has been demonstrated (Serganova I. et al., 2011). We hypothesize that tumor lactate levels monitored by MRSI will reflect LDH-A enzymatic activity, tumor “aggressiveness” and the propensity for developing metastases. The current study employs 4T1 cells bearing the stable produced LDH-A shRNA to down regulate the level of LDH-A expression. LDH-A silenced and control 4T1 breast cancer cells were injected into the mammary fat pad of athymic nu/nu female mice (5 mice for each group). The in vivo growth rate of the LDH-A silenced tumors was significantly slower than that of control tumors; the average doubling time of the LDH-A silenced tumors was 1.5 fold higher than control tumors (p<0.01). The lactate signal was acquired using a selective multiple-quantum coherence transfer (SelMQC) editing sequence in combination with chemical shift imaging (CSI) (He Q. et al., 1995). The tumors were scanned at small (∼100 mm3) and larger (∼300 mm3) volumes and in vivo lactate concentration was measured. The LDH-A silenced tumors produce less lactate, ∼ 6 mM at small volumes and ∼ 3 mM at larger volumes, whereas the control tumors produce ∼ 11 mM at small, and ∼7 mM at larger volumes. Preliminary results suggest that high tumor lactate concentrations are linked with a greater propensity to develop metastases. Studies evaluating metastatic burden are ongoing. We suggest that LDH-A expression and tumor lactate levels may be potential markers of elevated metastatic potential. In addition, MRSI might offer a noninvasive approach for assessing tumor progression and metastatic potential.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3223. doi:1538-7445.AM2012-3223
Abstract 3230: Fenofibrate-mediated energy crisis and apoptotic cell death of glioblastoma
Authors: Wilk Anna M. , Urbanska Katarzyna , Impastato David , Reiss Krzysztof
Published: August 2012

Glioblastomas are one of the most aggressive brain tumors which are characterized by rapid cell growth, infiltration of the Central Nervous System (CNS), and resistance to all known anticancer regiments. Therefore, the development of new more effective therapies against Glioblastoma is the major challenge for modern medicine. Fenofibrate, a drug widely utilized to lower lipid concentration in the blood, is one of the very promising anti-cancer treatments with low systemic toxicity. This potent agonist of PPARδ is thought to shift energy metabolism from glycolysis to fatty acid α-oxidation and, therefore, it may be toxic to glycolysis -dependent brain tumor cells (Warburg effect). While the toxicity to cancer cells appears to be clear, the precise mechanism/s of fenofibrate action against brain tumors seems to be much more complex and remains to be elucidated. To understand metabolic basis of the anti-cancer action of fenofibrate, we have analyzed extracellular acidification (ECAR) and oxygen consumption (OCR) using XF 24 Extracellular Flux Analyzer (Seahorse Bioscience), mitochondrial membrane potential as well as ATP level in different glioblastoma cell lines (LN229, U87MG, T98G). Surprisingly, we found that basal oxygen consumption (OCR) decreased significantly in Glioblastoma cells pretreated with fenofibrate, and the addition of metabolic toxins did not change their metabolic profile. Decreased oxygen consumption was due to the direct interaction of fenofibrate with complex I, a protein involved in the mitochondrial electron transport chain. We additionally found that, in parallel to the metabolic changes, fenofibrate triggers cell cycle arrest followed by apoptosis. The mechanisms involve a PPARδ-mediated effect, which leads to the energy crisis and apoptosis, as well as a PPARδ-independent pathway which results in FOXO3a nuclear translocation and activation of the proapoptotic protein Bim. Overall our data strongly support an anticancer action of fenofibrate by shifting the glycolitic-dependent cell metabolism to fatty acids α-oxidation with subsequent energy crisis and apoptotic cell death.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3230. doi:1538-7445.AM2012-3230
Abstract 3232: CZ48 activation by human tumor cytosol inhibited in the presence of NADPH
Authors: Markides Constantine S.A. , Battershell Lauren E. , Vardeman Dana M. , Giovanella Beppino C.
Published: August 2012

CZ48, the 20-O-propionate ester of Camptothecin (CPT), is a non-toxic prodrug of CPT first described by Cao et al. (1998). The propionate side-chain is enzymatically cleaved in target tissues. This gives rise to CPT, an inhibitor of Topoisomerase I, which then blocks cell division causing selective tumor toxicity. We have previously established the preferential activation of CZ48 in tumor tissues compared to normal tissues and an ultra-sensitive assay to detect it (2009). The present studies explore the specifics of that activation in vitro using both cells in culture and surgically removed xenografted tumors from nude mice. DOY lung adenocarcinoma was grown subcutaneously in nude mice and then surgically excised, homogenized and serially centrifuged to produce specific subcellular fractions. The bulk of esterase activity was found to be concentrated in the cytosol, while the microsomal fraction showed minimal activity. All other fractions tested were below the limit of detection. Microsomes showed only a fraction of the esterase activity of the cytosol (99.78 ± 14.89 ng/ml versus 487.48 ± 16.69 ng/ml CPT produced overnight), but even that little activity was eradicated in the presence of an NADPH redux regeneration system (10.0 ± 7.26 ng/ml). This inhibition was also observed in the cytosolic fractions. Deconstructing the redux system led to the conclusion that NADPH was inhibiting the activation of CZ48: when incubating cytosol with CZ48 in the presence of 26.1 mM NADPH, 66mM MgCl2, or 66mM Glucose-6-phosphate, only NADPH exhibited any effect on the esterase activity of CZ48, resulting in only 106.00 ± 15.49 ng/ml CPT produced overnight. These data reinforce the notion that CZ48 is metabolized to CPT by a non-microsomal, non CYP450 enzymatic process in tumor tissues.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3232. doi:1538-7445.AM2012-3232
Abstract 3237: XRay structure of human glutaminase in complex with BPTES and analysis of BPTES-resistant mutants
Authors: Delabarre Byron , Hurov Jonathan , Gross Stefan
Published: August 2012

Glutaminase (GLS1/2) catalyzes the conversion of L-glutamine to L-glutamate and ammonia. A splice variant of GLS1 (GAC) is elevated in certain cancers and is specifically inhibited by bis-2-(5-phenylacetimido-1,2,4, thiadiazol-2-yl)ethyl sulfide (BPTES). We describe here the first full length crystal structure of GAC both in the presence and absence of BPTES molecules. Two BPTES molecules bind at a interface region of the GAC tetramer in a manner which appears to lock the GAC tetramer into a non-productive conformation. The importance of these loops with regards to overall enzymatic activity of the tetramer was revealed by a series of GAC point mutants designed to create a BPTES resistant GAC. An unintended but nonetheless interesting transfer of phosphate activation activity was observed upon inserting key residues from GLS-2 into GLS-1.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3237. doi:1538-7445.AM2012-3237
Abstract 3242: Wnt secretion is required for colon cancer cell survival
Authors: Voloshanenko Oksana , Erdmann Gerrit , Iris Augustin , Boutros Michael
Published: August 2012

Aberrant activation of Wnt signalling has an important function in the onset and progression of colorectal cancer. Sporadic colorectal carcinomas are associated with mutations in APC or β-catenin, leading to a constitutive activation by preventing the degradation of β-catenin, allowing its accumulation in the nucleus and drive the expression of transcriptional target for growth and proliferation. While mutations in APC or β-catenin are found in almost all cases of sporadic colon cancer, the molecular circuits that control Wnt signalling pathways in colon cancer cells have not been fully resolved. Here we show that colon cancer cells require Wnt secretion for survival. We combined RNAi-mediated depletion of signalling factors and transcriptional profiling to map the topology of Wnt pathways. We demonstrate that Evi/Wls, a protein required for the secretion of Wnt proteins, impairs the expression of β-catenin dependent transcriptional targets. Blocking Wnt secretion is sufficient to inhibit Wnt signalling and cell survival of colon cancer cell lines ex vivo and in vivo, independent of their APC and β-catenin mutational status. We conclude that mutations in APC and β-catenin in colon cancer do not render colon cancers independent of autocrine activation through Wnt ligands. These results may open new avenues for therapeutic interventions in colon cancer by antagonists of Wnt signalling that act at or upstream of the receptor level.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3242. doi:1538-7445.AM2012-3242
Abstract 3246: TMS1 / ASC is critical for primary ciliogenesis and participates in cell migration
Authors: Abreu Eladio , Patel Pritty , Kline Erik , Marcus Adam , Vertino Paula
Published: August 2012

TMS1/ ASC encodes a bipartite signaling protein that functions in programmed cell death and proinflammatory response. Loss of function of TMS1/ASC through epigenetic silencing has been implicated in the pathogenesis of several malignancies, including breast and prostate cancer, although the precise mechanism by which TMS1 loss promotes tumorigenesis remains unclear. Recently, we showed that, in breast epithelial cells, TMS1/ASC silencing promotes resistance to detachment induced apoptosis or ‘anoikis’ through a mechanism that involves the inhibition of procaspase-8 cleavage and Bim upregulation. Here, we further characterized the role of TMS1 in breast epithelial cell adhesion and motility. We found that while TMS1 was dispensable for adhesion to fibronectin, it was necessary for efficient migration across a transwell chamber towards a chemoattractant and in wound-closure. siRNA mediated silencing of TMS1 inhibited breast cell invasion through a Matrigel matrix. Subcellular localization studies showed that adhesion of breast epithelial cells to a fibronectin substratum induced the aggregation of TMS1 into barrel-like structures that formed at the apical cell surface. Primary cilia are solitary, sensory organelles that serve as platforms for multiple signaling pathways. Signalling from these organelles plays an important role in linking environmental cues to cell growth control. Interestingly, primary cilia are observed at much lower frequencies on cancer cells. Physical and temporal similarities between the observed TMS1 barrel-like structures and the basal structures of primary cilia suggest that TMS1 may be linked to adhesion and migration activities by regulating the function and/or biogenesis of primary cilia. To test this hypothesis, we used HEK293 kidney epithelial cells expressing an inducible myc-tagged TMS1 and TMS1-/- MEFs to explore the role of TMS1 in primary ciliogenesis. We found that in the absence of TMS1, HEK293 cells were unable to produce primary cilia, but cilia formation could be induced upon TMS1 induction. Taken together, our studies provide evidence of a novel non-apoptotic, inflammasome-independent role for TMS1 in epithelial cell biology. Furthermore, TMS1 silencing may contribute to carcinogenesis by conferring a tumor suppression disadvantage resulting from primary ciliogenesis deficiencies. Supported by Department of Defense/CDMRP Breast cancer grant W81XWH-08-1-0390 and NIH Institutional Research and Academic Career Development Award (IRACDA) K12GM000680.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3246. doi:1538-7445.AM2012-3246
Abstract 3383: Expression and role of BORIS/CTCFL in human cancer stem cells
Authors: Benhattar Jean , Alberti Loredana
Published: August 2012

The existence of CSCs, a small population that drives the tumors development, progression, metastasis and drug resistance, could explain the failure of several anti-cancer therapy. The discovery of the genes responsible for the CSCs phenotype would point out to the development of new target for the cancer therapy. To date, many tumor markers have been identified but almost none of them are specific to target the CSCs population. The cancer testis gene BORIS/CTCFL was shown to be exclusively expressed in germ cells in normal tissues and was involved in epigenetic reprogramming. BORIS is slightly activated in a wide variety of tumors. It is not clear if this expression is due to basal expression in almost all tumor cells or rather to the expression in a restricted population of cells, as in the CSC. To date, there is no validated antibody against BORIS. Therefore, to analyze BORIS expression in vivo, we developed a molecular beacon (MB) imaging technology to detect BORIS mRNA gene expression. BORIS MB binds to its specific target and produced fluorescence signal at 37°C in vitro, as well as in vivo in the cells expressing high level of BORIS mRNA. BORIS was found to be expressed only in Side Population cells (Hoechst negative cells) in different tumor cell lines (HeLa, HT29, Raji, MB-MDA-231). Our data indicates that BORIS-positive cells are present at low frequency (0.01% is the estimated frequency) in tumor cell lines. Quantitative RT-PCR analysis revealed a significantly higher expression level of BORIS in SP sorted cells (10-20 fold) compared to that from non-SP cells, similar resultswere obtained in colon spheres (40 fold) compared to adherent cells. This result indicates that BORIS is overexpressed in CSCs enriched population of tumor cells. To define the potential role of BORIS in CSCs, we performed shRNA knockdown experiments in several tumor cell lines. We found that BORIS mRNA inhibition could allow decreasing cancer cell survival in studied tumor cell lines, supporting the hypothesis that BORIS could be critical for the survival and self-renewal of tumors. In conclusion, our study provides evidence that BORIS/CTCFL is a promising marker of CSCs that could also play a critical role in the maintenance of self-renewal capacity of the tumor initiating cells and as a consequence, of tumor survival. Therefore, due to its restricted expression in normal tissues and its reexpression in a wide variety of tumors, BORIS/CTCFL could become a good target for cancer therapy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3383. doi:1538-7445.AM2012-3383
Abstract 3391: Interaction of microRNAs and target genes in bladder cancer metastasis
Authors: Ru Yuanbin , Kechris Katerina J. , Smith Steven C. , Calin George A. , Theodorescu Dan
Published: August 2012

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression through base pairing with other mRNA transcripts. MiRNA expression signatures have characterized many phenotypes in bladder cancer, including grade, stage, and metastasis. However, simultaneous mRNA changes accompanying the miRNA profile, along with the miRNA:mRNA interaction, were not systematically studied in these phenotypes. To gain insights into the miRNA regulatory network in bladder cancer metastasis, we profiled miRNA and mRNA expressions in two pairs of metastatic and non-metastatic cell lines. Interactions between differentially expressed miRNAs and mRNAs were predicted using six software. We identified 194 miRNA:mRNA pairs between 18 up-regulated miRNAs and 66 down-regulated genes in metastatic cells; while the down-regulation of miRNAs and the increased expression of target genes were rare. The overexpression of one of the miRNAs, hsa-miR-146a, is confirmed by quantitative RT-PCR in metastatic cell lines. The 66 potential target genes are enriched in cytoskeleton organization and actin binding, underlining the importance of cell motility in cancer metastasis. Analysis of microarray data from two patient cohorts indicated lower expression of the 66 miRNA target genes in tumors than in normal bladder tissues. Our results suggest the significance of miRNA regulatory network in cancer metastasis and provide a list of potential miRNA:mRNA pairs for further investigation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3391. doi:1538-7445.AM2012-3391
Abstract 3413: A population of metastatic colorectal cancer (mCRC) patients with radiologically proven liver metastasis and EpCAM expressing primary tumors show low circulating tumor cell counts by Veridex Cellsearch
Authors: Das Avisnata , Joudeh Jamal , Peters Kristi L. , Allen Joshua E. , Zheng Siyang , Dicker David T. , Yang Zhaohai , El-Deiry Wafik S.
Published: August 2012

Circulating tumor cells (CTCs) have prognostic significance for metastatic cancers including colorectal, breast and prostate cancer. We have been evaluating CTC numbers as well as molecular markers as prognostic, treatment and disease-monitoring tools. The FDA recommends a cutoff value of > 3 CTCs/7.5 ml of blood, using Johnson & Johnson Veridex Cellsearch technology as indicator of poor prognosis for metastatic colorectal cancer (mCRC). We report identifying a population of patients who have advanced disease and liver metastases (shown by CT/MRI/PET scan) but the CTC count using our CLIA-certified Veridex Cellsearch laboratory was <3/7.5 ml of blood. As Cellsearch technology relies on Epithelial Cell Adhesion Molecule (EpCAM) expression for isolation of CTCs, we analysed the primary tumors for EpCAM positivity using immunostaining. All 5 patients we have identified so far showed moderate to strong EpCAM expression in their primary tumor. The low CTC count in these patients with reference to the FDA standard raises a question regarding use of CTC counts as an independent tool for measuring advanced disease as liver metastasis signifies progressive disease and is generally associated with poorer outcome. The epithelial-to-mesenchymal transition (EMT) in tumor cells in blood may provide a possible mechanism for loss of EpCAM expression in some CTCs although we have certainly detected similar patients with liver metastases from CRC with elevated levels of CTCs including about 10-15% of such CRC patients with very high levels at ∼10-20 times the cut-off of 3 CTCs/7.5 ml of blood. Technologies that involve detection of cancer cell proteins and do not rely on EpCAM-based isolation (eg. multiplex PCR) are not currently approved by the FDA for clinical application but may provide insights into this problem. Alternatively we are using microfluidic devices, that rely on size-based capture of CTCs in attempts to determine if such patients with liver metastases and no detectable CTCs by Veridex may have tumor cells that may be captured. We are particularly interested in determining whether the biology of the tumors varies among patients with liver metastases from CRC with no measurable CTCs versus those patients with high levels of CTCs. In this regard we are pursuing molecular characterization of primary tumors looking for possible molecular insights among these cohorts.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3413. doi:1538-7445.AM2012-3413
Abstract 3416: Ubiquitous Brms1 expression is critical for mammary carcinoma metastasis suppression via promotion of apoptosis
Authors: Cook Leah M. , Cao Xuemei , Dowell Alexander E. , Frost Andra A. , Desmond Renee A. , Edmonds Mick D. , Kesterson Robert A. , Hurst Douglas R. , Welch Danny R.
Published: August 2012

Morbidity and mortality in breast cancer patients are drastically increased when primary tumor cells are able to spread to distant sites and proliferate to become secondary lesions. Effective treatment of metastatic disease has been limited; therefore, increased molecular understanding to identify biomarkers and targets is needed. We have previously shown that breast cancer metastasis suppressor 1 (Brms1) can suppress development of pulmonary metastases when expressed in a variety of cancer types, including metastatic mammary carcinoma. Our lab has developed two transgenic Brms1 mouse models, one which expresses murine Brms1 cDNA specifically in mammary tissue (expression by the mouse mammary tumor virus (MMTV) promoter) and a ubiquitous Brms1 expression model (expression by the chicken beta actin promoter). The goal of this study was to investigate mechanisms of Brms1-mediated metastasis suppression in transgenic mice that express Brms1 using a polyoma middle T (PyMT) oncogene-induced model. Brms1 expression, either ubiquitously or predominantly in the mammary gland, did not significantly alter growth of the primary tumor, confirming earlier studies. When expressed ubiquitously, Brms1 suppressed pulmonary metastasis and promoted tumor cell apoptosis in the lung but not in the mammary gland. However, selective expression of Brms1 in the mammary gland using the MMTV promoter did not significantly block metastasis nor did it promote apoptosis in mammary glands or lungs despite increased expression within primary tumors and the lungs. These results suggest tissue- or cell-type specific expression of Brms1 is a critical determinant for Brms1-mediated metastasis suppression.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3416. doi:1538-7445.AM2012-3416