bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3941: HAF promotes the switch from HIF-1α to HIF-2α signaling during chronic hypoxia and promotes stem cell characteristics
Authors: Koh Mei Yee , Lemos Robert , Powis Garth
Published: August 2012

Most solid tumors and their metastases experience periods of hypoxia, which promotes tumor progression and resistance to therapy. Hypoxia is also an important micro-environmental factor supporting the maintenance of pluripotent cancer stem/progenitor cells (CSCs) within the stem cell niche. The clinical relevance of CSCs is underscored by their resistance to cytotoxic therapies, invasive potential and role in tumor recurrence. Critical mediators of the hypoxia response are the hypoxia-inducible factors HIF-1α and HIF-2α. Although they have some overlapping functions, the HIFs are generally non-redundant whereby HIF-1α regulates the acute metabolic changes required for hypoxic survival, whereas HIF-2α promotes the adaptation to chronic hypoxia, including the maintenance of CSCs. However, the mechanism determining selectivity for HIF-1α versus HIF-2α activation is unclear. Here we describe a new mechanism for the switch between HIF-1α and HIF-2α dependent transcription during chronic hypoxia by the hypoxia associated factor (HAF). HAF is overexpressed in a number of tumor types and is an E3 ubiquitin ligase that ubiquitinates HIF-1α independently of oxygen and pVHL, thus targeting HIF-1α for proteasomal degradation. HAF also binds to HIF-2α but at a different site than it binds to HIF-1α, increasing HIF-2α transactivation without causing its degradation. Hence HAF switches cellular dependence from HIF-1α towards HIF-2α, resulting in the activation of HIF-2α specific target genes (MMP9, OCT3/4)and the simultaneous inhibition of HIF-1α specific target genes (CA-IX, DDIT4). HAF itself is hypoxia regulated - decreasing during acute hypoxia, but increasing with hypoxic duration. Hence HAF is a key determinant of HIF-2 versus HIF-1 activation in response to hypoxia. Using cell-free recombinant proteins and in vitro binding assays, we show that HAF binding to HIF-2α requires a novel eukaryotic post-translational modification of HIF-2α within a unique region of HIF-2 that is absent from HIF-1. HAF binding to HIF-2α is increased in cells exposed to prolonged hypoxia and promotes the transcription of HIF-2α target genes important in CSC maintenance such as OCT3/4 and SOX2. HAF levels are elevated in CD133+ U87 glioblastoma stem/progenitor cells and HAF knockdown decreases neurosphere formation suggesting a key role for HAF in the maintenance of the stem cell phenotype. The HAF-induced switch to HIF-2α dependent transcription promotes invasion and enriches the proportion of stem/progenitor CD133+ U87 cells. This increases the rate of tumor take and increases tumor morbidity in vivo. Funded by NIH CA095060, CA17094, CA098920

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 3941. doi:1538-7445.AM2012-3941
Abstract 3949: In vivo imaging of tumor hypoxia by a new near-infrared fluorescent carbonic anhydrase IX-targeted agent
Authors: Bao Bagna , Groves Kevin , Zhang Jun , Handy Emma , Kennedy Paul , Cuneo Garry , Supuran Claudiu T. , Yared Wael , Rajopadhye Milind , Peterson Jeffrey D.
Published: August 2012

Carbonic anhydrase IX (CA IX), a cell surface enzyme involved in tumor cell acidification, is induced during tumor hypoxia (lack of oxygen) in many different types of tumors. CA IX expression is correlated to tumor cell proliferation and metastasis, poor prognosis, and resistance to therapeutic intervention, making CA IX an important biomarker in the study of hypoxia, tumor cell proliferation, and therapy. Therefore, determination of CAIX expression and identification of hypoxic tumors in vivo are critical for cancer research and treatments. We have designed and synthesized a near-infrared (NIR) fluorescent agent (VM3219) for in vivo detection and quantification of a cell surface biomarker of hypoxia, CA IX based on a well-known CA IX inhibitor, acetazolamide. VM3219 was characterized extensively in HeLa cells and in mouse HeLa tumor xenografts. Preliminary chemical and biochemical characterization of VM3219 showed excellent CA IX selectivity and affinity. In vitro, VM3219 detected 4-7-fold up-regulation of CA IX in live hypoxic HeLa cells as visualized by fluorescence microscopy and quantified by flow cytometry. VM3219 fluorescence signals were blocked by 80% when cells were pre-incubated with unlabeled acetazolamide. Fluorescent signal of VM3219 was low in normoxic HeLa cells and was comparable to the signal that were detected by using a non-binding control agent (VM3182) incubated with normoxic or hypoxic cells. In vivo imaging by fluorescence molecular tomography (FMTTM) showed that approximately 5% of the injected dose (2 nmol VM3219) was retained in the tumor tissues after 24 h (100 -120 pmol), 10-20-fold more than measured with a non-binding control agent. Tumor fluorescence was blocked >65% by prophylactic acetazolamide treatment. Maintenance of tumor-bearing mice on low oxygen (8%), as compared to normally housed tumor bearing mice (20% oxygen), induced a >2X (∼250 pmol) increase in VM3219 tumor fluorescence signal suggesting that CAIX expression was up-regulated within tumors by low oxygen. In tissue sections, VM3219 signal was shown to be specifically co-localized in the tumor hypoxic regions with CA IX antibody and Hypoxyprobe (pimonidazole), and the signal was away from non-hypoxic regions as determined by Hoechst stain. In conclusion, VM3219 is a new non-invasive tumor hypoxia imaging agent that offers a tool for specific imaging of a biologically important hypoxia biomarker, CA IX.

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 3949. doi:1538-7445.AM2012-3949
Abstract 3966: GenomeSpace: An environment for frictionless bioinformatics
Authors: Reich Michael , Liefeld John , Thorvaldsdottir Helga , Ocana Marco , Polk Eliot , Jang DK , Mesirov Jill
Published: August 2012

Over the past several years, cancer genome characterization initiatives such as The Cancer Genome Atlas and the Tumor Sequencing Project have produced an explosion of genomic data. The pace of data production has recently increased with the adoption of next-generation sequencing technologies and large-scale data production efforts to discover the breadth of genomic variation in humans. Comprehensive analysis of these datasets requires the coordinated use of Web-based applications and data repositories, desktop analysis tools and visualizers, and single-purpose algorithms. However, the effort required to transfer data between tools, convert between data formats, and manage results often prevents researchers from utilizing the wealth of methods available to them. Many integrative genomics and translational “bench to bedside” discoveries are possible with combinations of existing tools, but the necessary transitions between them puts them out of the reach of most researchers. GenomeSpace, http://www.genomespace.org, is an environment that brings together diverse computational tools, enabling scientists without programming skills to easily combine their capabilities. It aims to offer a common space to create, manipulate and share an ever-growing range of genomic analysis tools. GenomeSpace features support for cloud-based data storage and analysis, multi-tool analytic workflows, automatic conversion of data formats, and ease of connecting new tools to the environment. A set of six “GenomeSpace-enabled” seed tools developed by collaborating organizations provides a comprehensive platform for the analysis of cancer data: Cytoscape (UCSD), Galaxy (Penn State University), GenePattern (Broad Institute), Genomica (Weitzmann Institute), Integrative Genomics Viewer (Broad Institute), and the UCSC Genome Browser (UCSC). We show how researchers can use GenomeSpace to effortlessly combine the capabilities of all of these tools in several cancer research scenarios.

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 3966. doi:1538-7445.AM2012-3966
Abstract 4250: Deficiency of CD109, a negative regulator of TGF-β signaling, leads epidermal hyperplasia and appendage abnormalities in mice
Authors: Mii Shinji , Murakumo Yoshiki , Asai Naoya , Asai Masato , Takahashi Masahide
Published: August 2012

CD109, a glycosylphosphatidylinositol (GPI)-anchored glycoprotein, is a member of the β2-macroglobulin/C3, C4, C5 family of thioester-containing proteins. It has been reported that CD109 is highly expressed in various tumor cell lines and in tumor tissues including squamous cell carcinomas (SCCs) of the lung, oral cavity, esophagus and uterine cervix, malignant melanoma of the skin, and urothelial carcinoma of the urinary bladder. CD109 is, therefore, thought to be a cancer-related gene. On the other hand, CD109 is a component of TGF-β receptor system and negatively regulates TGF-β signaling. However, its physiological function in vivo remains largely unknown. To investigate the physiological function of CD109 in vivo, we generated CD109-deficient (CD109-/-) mice in which exon 1 and 2 of CD109 genomic locus were replaced with the lacZ gene. CD109 protein is expressed in the skin, tongue, and testis of adult CD109+/+ mice, but not in CD109-/- mice. Macroscopically, CD109-/- mice showed hair growth impairment from postnatal day 7 (P7) and persisted until P28. Microscopically, hyperplasia of the epidermis and sebaceous glands became apparent at P7 in CD109-/- mice and sustained until P70, and the hair follicles of CD109-/- mice exhibited ectasia with kinked hair shafts at P14. The epidermis of CD109-/- mice showed apparent thickening of the basal / suprabasal layers, which are positive for CK14, compared with CD109+/+ mice, whereas no significant differences in Ki-67 or cleaved caspase-3-positive ratios were detected between the two groups. These results suggest that CD109 regulates the differentiation of keratinocytes in vivo, and impairment of hair growth and ectatic hair follicles may be secondary changes caused by narrowing of the infundibular portion of the hair follicles due to epithelial hyperplasia.

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 4250. doi:1538-7445.AM2012-4250
Abstract 4276: PGE2 induces LEF-1 expression in breast cancer cells, but is not required for the nuclear localization of beta-catenin
Authors: Stasinopoulos Ioannis , Greenwood Tiffany , Glunde Kristine , Bhujwalla Zaver M.
Published: August 2012

We have previously demonstrated that poorly differentiated MDA-MB-231 cells displayed delayed tumor onset, reduced frequency of occurrence and loss of their capacity for extrapulmonary colonization when transfected stably with a potent and specific cyclooxygenase (COX) −2 shRNA sequence. We attributed the reduction in tumor growth and extrapulmonary colonization to reduced invasive capabilities, reduced angiogenesis, altered metabolism and differences in the stabilization of hypoxia-inducible factor-1α. We also observed loss of expression of oncogenes, receptors and degradative enzymes associated with tumor growth and metastasis including matrix metalloproteinase-1, CXC Receptor-4, roundabout-1, hyaluronan synthase-2, and monocarboxylate transporter 2. In addition to these previously reported transcriptome changes, we observed that the most highly downregulated transcription factor was Lymphoid-Enhancer Factor-1 (LEF-1) a result validated by qPCR showing an 8-fold loss of LEF-1 in COX-2-silenced cells. Here, we present evidence demonstrating that LEF-1 accumulation in the nucleus of COX-2-silenced cells is reduced compared to COX-2 containing parental cells. Induction of COX-2 by phorbol esters resulted in nuclear accumulation of LEF-1 in COX-2 containing, but not in COX-2-silenced, cells. Addition of physiological concentrations of PGE2 to COX-2-silenced cells partially restored LEF-1 expression in the nucleus of these cells. LEF-1 is a member of the T-cell specific factor (TCF) family of transcription factors that direct transcription of downstream target genes upon nuclear translocation of β-catenin from the cytoplasm. β-catenin translocation to the nucleus occurs in response to canonical signaling of the ligand Wnt to its receptor Frizzled. An earlier study showed that the COX-2 product prostaglandin E2 promoted the translocation of β-catenin to the nucleus of colon cancer cells via G-protein coupled receptor signaling. As expected, immunofluorescence of COX-2 containing MDA-MB-231 breast cancer cells showed nuclear expression of β-catenin. However, the nuclear localization of β-catenin persisted even in the absence of PGE2, as observed in COX-2-silenced cells, suggesting that PGE2 expression is not necessary for the nuclear localization of β-catenin in MDA-MB-231 cells. Taken together, our results demonstrate that while PGE2 is not necessary for the nuclear localization of β-catenin in MDA-MB-231 cells, it can promote the nuclear accumulation of the TCF family member LEF-1, thereby further promoting the non-canonical activation of the Wnt pathway in breast 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 4276. doi:1538-7445.AM2012-4276
Abstract 4294: GE152: In vivo detection of tumor apoptosis as a tool for assessment of therapeutic efficacy
Authors: Hiscock Duncan R. R. , McRobbie Graeme , Lear Rochelle E. , Danikas Antonios , Cawthorne Chris , Simpson Kathryn , Zivanovic Maureen , Brown Gavin , Price Andrew , Williams Kaye J. , Dive Caroline
Published: August 2012

A major challenge in personalized healthcare is predicting how effective a drug treatment is. This is particularly the case in oncology, where there is a call for better therapy monitoring to maximize drug treatment effectiveness leading to improved patient survival and help reduce healthcare costs. GE152, a nuclear imaging agent under development at GE Healthcare, is being evaluated preclinically as a tool to assess therapeutic efficacy by detecting tumor apoptosis. GE152 is based on a 99mTechnetium radiolabelled peptide that shows nanomolar affinity for a specific cell death target, as demonstrated by studies using Biacore technology. Biodistribution using the murine lymphoma (EL4) tumour therapy model has shown increased tumor uptake and retention of GE152 following chemotherapy, with positive tumor:muscle and tumor:blood ratios. Correlation of tumor apoptosis levels (determined by caspase activity) with GE152 tumor retention suggest a trend of increasing agent retention with rising levels of apoptosis (GE152 retention in low apoptotic tumors is 4.9%ID/g; GE152 retention in high apoptotic tumors is 8.2%ID/g).Further validation of GE152 was carried out using an apoptosis-specific inducible cell death model whereby HT29 colorectal cancer cell xenografts engineered to inducibly express either a constitutively active form of caspase-3 (which causes synchronous cell death in vivo when exposed to doxycycline) or an inactive point mutant. GE152 demonstrated greater uptake in tumors undergoing apoptosis (3.6% ID/g) than in controls (1.2% ID/g), correlating with caspase activity levels (and subsequent apoptosis) as determined both enzymatically and by IHC and blood-borne biomarkers of cell death. These results are in agreement with preliminary preclinical imaging studies using SPECT/CT, where region of interest analysis has demonstrated increased post-therapy tumor retention of GE152. We are currently optimizing the performance of GE152 by assessing different radiolabelling precursors to improve imaging agent pharmacokinetics. In addition to the current 99mTechnetium-based approach, we are exploring 18F radiolabelling options that would allow expansion of the agent's utility to PET imaging.

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 4294. doi:1538-7445.AM2012-4294
Abstract 4316: Villin expression is frequently lost in colon cancers with microsatellite instability.
Authors: Arango Diego , Al-Obaidi Sheren , Williams David S. , Dopeso Jose , Mazzolini Rocco , Corner Georgia A. , Byun Do-Sun , Murone Carmel , Togel Lars , Zeps Nikolajs , Aaltonen Lauri A. , Iacopetta Barry , Mariadason John Martin
Published: August 2012

Colorectal cancers are classified as having chromosomal instability (CIN) or microsatellite instability (MSI). MSI colon cancers frequently display poorly differentiated histology the molecular basis of which is not well understood. Gene expression profiling of CIN and MSI colon cancer cell lines and tumours revealed significant downregulation of the intestinal-specific cytoskeletal protein villin, in MSI tumours, with complete absence observed in 62% and 17% of MSI cell lines and primary tumours, respectively. Investigation of 577 colon cancers demonstrated loss of villin expression was linked to poorly differentiated histology in MSI and MSS (microsatellite stable) tumours. Furthermore, mislocalization of villin away from the membrane was prognostic for poorer outcome in MSS patients. Loss of villin expression was not due to coding sequence mutations, epigenetic inactivation, or promoter mutation. Conversely, villin promoter activity reflected endogenous villin expression, suggesting villin loss is transcriptionally mediated. A screen of gut-specific transcription factors revealed a significant correlation between expression of villin and the homeobox transcription factor, Cdx-1. Cdx-1 overexpression induced villin promoter activity, Cdx-1 knockdown downregulated endogenous villin expression, and deletion of a key Cdx binding site within the villin promoter attenuated promoter activity. Loss of Cdx-1 expression was associated with Cdx-1 promoter methylation. These findings demonstrate that loss of villin expression as a result of Cdx-1-dependent transcriptional deregulation is a feature of poorly differentiated colon 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 4316. doi:1538-7445.AM2012-4316
Abstract 4340: Increased cytokine and chemokine expression in U87MG glioblastoma cells after large clinically relevant single doses of ionizing radiation
Authors: Mcdonald J. Tyson , Lee Jawon , Rietman Edward , Lamont Clare , Enderling Heiko , Peluso Michael , Hlatky Lynn
Published: August 2012

BACKGROUND: Stereotactic radiosurgery is an established treatment option that uses high, single fraction doses of radiation to target tumors. Despite the best clinical strategies to eliminate brain tumors using this technique or others (surgery, chemotherapy and/or other radiotherapy techniques), post-therapy recurrence of brain tumors remains a major challenge in patients. To better understand the radiation response, we investigated how such high doses modulate the molecular signaling within glioblastoma cell populations from several days to months after irradiation. METHODS: The glioblastoma cell line, U87MG, was treated with single doses of ionizing radiation between 0 to 16Gy in vitro followed by: examination of cell proliferation; clonogenic survival; extraction of RNA; and subcutaneous injection into nude mice to monitor tumor growth 24 hours after cell irradiation. Genome-wide expression profiling was run on the Illumina HT-12 bead array platform and analysis for significant expressed genes was performed along with network analysis using Ingenuity Pathway Analysis and GenePattern platforms. RESULTS: As expected, high doses of radiation stunted growth of the U87MG cells from 0 to 14 days. Long term, single cell clonogenic survival of the irradiated cells was approximately 3% and 0.01% following 8Gy or 16Gy, respectively. Genome-wide gene expression at 1, 4 and 6 days after irradiation of 8 or 16Gy has revealed a dose-dependent increase in inflammatory cytokines and chemokines. Increased cytokine expression for IL-6 and IL-8 was validated in vitro using qRT-PCR. In vivo, 100% of the untreated U87MG cells formed tumors, whereas less than half of all irradiated treatment groups failed to form tumors. In those samples that did grow despite irradiation, the average time to tumor formation (volume = 300mm3) was 13, 32, 58 and 88 days for 0, 8, 12, and 16Gy respectively while the exponential fits to tumor growth rate was shown to be accelerated after 8Gy but diminished after 12 and 16Gy. IL-6 gene expression was found to be elevated in tumors formed from irradiated cells versus control. CONCLUSION: The data presented here provides novel insight into the cellular and molecular modulation of glioblastoma cell populations by large, clinically relevant, single doses of ionizing radiation. A salient finding is the expression of inflammatory cytokines as a major player in the radiation response at periods long after the irradiation insult. This study was supported by NIH ICBP 1U54CA149233

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 4340. doi:1538-7445.AM2012-4340
Abstract 4357: The role of lysyl oxidase on proangiogenic phenotypes of tumor endothelial cells
Authors: Osawa Takahiro , Ohga Noritaka , Hida Yasuhiro , Kitayama Kazuko , Yamamoto Kazuyuki , Kawamoto Taisuke , Maishi Nako , Kondo Miyako , Akiyama Kosuke , Onodera Yuichiro , Shinohara Nobuo , Nonomura Katsuya , Shindoh Masanobu , Hida Kyoko
Published: August 2012

Introduction and Objectives: The molecules which are highly expressed in tumor endothelial cell (TEC) are important to target TEC more specifically. We found LOX gene was upregulated in TEC compared to normal endothelial cell (NEC) by DNA microarray analysis in three different types of human tumor xenografts in nude mouse including renal cell carcinoma (RCC). LOX is an enzyme which is reported to play a critical role in the metastatic potential of tumor, possibly promoting tumor cell trafficking into the premetastatic niche in various types of malignancies. However, the effect of LOX on TEC has not been reported. To investigate whether LOX might be a TEC-specific target in cancer therapy, we analyzed its expression in clinical sample and examined the function of LOX on TEC. Methods: We successfully purified human TEC (hTEC) and NEC (hNEC) from surgically resected RCC and normal parenchyma from six patients, respectively. Using cultured hTEC and hNEC, competitive reverse transcription-polymerase chain reaction (RT-PCR) was performed. LOX expression level was investigated using immunohistochemistry (IHC). To explore the role of LOX in TEC, mouse TEC (mTEC) and NEC (mNEC) were isolated from human tumor xenografts in nude mouse and the normal counterparts, respectively. To investigate the function of LOX, LOX was knock-downed using siRNA and TEC phenotypic changes were analyzed by migration assay and vascular tube formation assay. In addition, we investigated the effect of α-aminopropionitrile (BAPN), an irreversible inhibitor of LOX, on tumor angiogenesis and metastatic colonization in vivo. Results: LOX expression levels were higher in isolated TEC than NEC both in human (Tissues: clear cell carcinoma 6; Stage T1, 3; T3, 3; Grade 1, 1; Grade 2, 2; Grade 3, 3) and mouse by RT-PCR. In IHC, LOX was detected in blood vessels in human RCC, not in normal tissue. Cell migration and tube formation of mTEC were suppressed by LOX knock-down using siRNA with decrease in phosphorylation of focal adhesion kinase (Tyr397). Furthermore, BAPN specifically inhibited tumor angiogenesis and micrometastasis in vivo, via inhibition of LOX activity of TEC. Conclusions: Our findings suggest that LOX upregulated in TEC contributes in their proangiogenic phenotype and plays key roles in tumor metastasis. LOX might be a TEC-specific target and be useful for cancer therapeutic purposes.

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 4357. doi:1538-7445.AM2012-4357
Abstract 4359: Use of high resolution Doppler imaging for non-invasive, quantitative evaluation of hypoxia-driven neovascularization following surgical ligation of the femoral artery
Authors: Duesbery Nick , Boguslawski Elissa , Merrifield Brittany , Chang Anthony , Chambers Christopher
Published: August 2012

A tumor's growth is inextricably linked with its vascularization. Rapidly growing tumors outstrip their blood supply, creating hypoxia. To compensate, tumor cells release angioproliferative factors that stimulate endothelial cell proliferation. Proliferating endothelial cells contribute to the formation of new blood vessels that promote tumor perfusion and stimulate further tumor growth. The close link between tumor growth and neovascularization makes it difficult to study either process in the absence of the other. To address this we have adapted a mouse model used in the study of peripheral arterial disease. Ischemia was induced by surgical ligation and excision a 3-5mm portion of the femoral artery, vein, and all nearby arterial branches in the right hindlimb of 10-12 week old athymic nude mice. Limb perfusion was measured prior to surgery and 1, 7, 14, 21, and 28 days post-surgery. A 7mm portion of hindlimb perfusion was quantified using Power Doppler mode on the VisualSonics Vevo 770 High Resolution micro Ultrasound system. Morphological changes were observed by histology and immunohistochemistry using antibodies against CD31 and F4/80. Based on quantified Doppler ultrasound data, limb perfusion was reduced as much as 80% within 24 hrs of ligation. Blood flow was restored over the course of the next 7 days. However, re-perfused limbs showed a diffuse pattern of blood flow in comparison to control limbs. Loss of perfusion was accompanied by muscle atrophy and the accumulation of intra-fasicular fat deposits. IHC revealed a marked increase of F4/80 and CD31 staining in ligated limbs. Similar changes were not observed in contralateral limbs that were not operated on or in sham-ligated hind limbs. Our data is consistent with the interpretation that femoral artery ligation causes acute loss of perfusion that is compensated for by macrophage recruitment and disseminated neovascularization. This model provides a novel opportunity to study neovascularization in the absence of tumor growth that will complement ongoing studies using tumor-based models of angiogenesis.

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 4359. doi:1538-7445.AM2012-4359
Abstract 436: Resistance to fluid shear stress is a conserved novel biophysical property of malignant cells
Authors: Nauseef Jones T. , Barnes J. Matthew , Henry Michael D.
Published: August 2012

During metastasis, cancer cells access distant tissues via the circulation but how this fluid microenvironment influences cancer cell biology is poorly understood. It has been suggested that physical destruction of carcinoma cells due to exposure to hemodynamic forces during circulation is a barrier to metastasis. Here we report that transformed cells are remarkably resistant to fluid shear stress (FSS) in a novel microfluidic protocol. When subjected to a series of millisecond pulses of high FSS, transformed cells exhibit a biphasic decrease in viability. Significantly, human red and white blood cells survived the forces in this protocol. In stark contrast, non-transformed epithelial cells are markedly more susceptible to death under FSS. Accordingly, we show that FSS resistance is a property conferred by oncogenes. We reveal FSS resistance to be a transient and adaptive response triggered by plasma membrane damage and mediated by both extracellular calcium influx and dynamic reorganization of the actin cytoskeleton. This property of malignant cancer cells may facilitate hematogenous metastasis and indicates, contrary to expectations, that cancer cells are quite resistant to FSS. Thus, destruction of circulating cancer cells by hemodynamic shear is not likely to limit metastasis. Our data suggest that FSS resistance is a biophysical biomarker which may be used to rapidly discriminate between malignant and benign cells and, by virtue of representing phenotypic integration of oncogenic transformation, may be more tractable than conventional molecular biomarkers.

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 436. doi:1538-7445.AM2012-436
Abstract 4362: Identification and isolation of host factors in response to therapy: an approach to identify new targets for cancer therapy
Authors: Gingis-Velitski Svetlana , Voloshin Tali , Fremder Ella , Shaked Yuval
Published: August 2012

Chemotherapy remains one of the most common treatment modalities for cancer, and while initial tumor regression is usually observed, tumor re-growth and metastatic spread sometimes occur. Several preclinical studies have shown that, under certain circumstances, chemotherapy may induce angiogenesis and metastases. Several mechanisms were suggested for explaining tumor recurrence following therapy, and they are mostly related to the acquired resistance of tumor cells to a specific therapy. A recent body of evidence suggests that host derived molecular and cellular mechanisms may also account for the pro-tumorigenic and pro-metastatic effects of tumor cells following chemotherapy, yet there is still ample necessity for further investigation and isolation of such host factors. In order to solely study the host-driven pro-tumorigenic effects of chemotherapy on tumor cells, we used non-tumor bearing mice which were treated with paclitaxel chemotherapy. Subsequently, when the cytotoxic drug was cleared from the circulation, plasma was collected and used in various screening technologies in order to isolate and identify possible factors which may alter the tumor microenvironment for the tumor's advantage. We found that factors promoting bone marrow derived cell mobilization and angiogenesis, e.g., stromal-derived factor 1-α (SDF-1α) and granulocyte colony-stimulating factor (G-CSF) are substantially upregulated in the plasma of paclitaxel treated mice. In addition, increased activity of metalloproteinase 2 (MMP2) and MMP9 were also observed in plasma and bone marrow derived cells collected from mice primed with paclitaxel chemotherapy. The inductions of such host factors contributed to tumor re-growth due, in part, to their role in angiogenesis mediated by bone marrow derived cells, and to their impact on tumor cell migration and invasion activity which can lead to metastases. The pharmacologic or genetic blockade of each of these factors in conjunction with paclitaxel chemotherapy increased the treatment outcome. Additional factors are currently being evaluated and validated with respect to their impact on tumor re-growth following therapy, and will be presented in the upcoming meeting. Collectively, the evaluation of upregulated host factors in response to chemotherapy may raise potential new targets for cancer therapy, and may therefore contribute to the development of new treatment approaches for prolonging the efficacy of conventional cancer 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 4362. doi:1538-7445.AM2012-4362
Abstract 4698: Inhibition of histone deacetylase (HDAC) 3 induces hyperacetylation and inhibition of nuclear heat shock protein (hsp) 90 leading to depletion of ATR and CHK1 with sensitization to DNA damage in breast and cervical cancer cells
Authors: Ha Kyungsoo , Fiskus Warren , Balusu Ramesh , Rao Rekha , Venkannagari Sreedhar , Bhalla Kapil N.
Published: August 2012

We have recently reported that in addition to checkpoint kinase 1 (CHK1), ataxia-telangiectasia mutated and Rad3- related (ATR) is chaperoned by hsp90. Treatment of breast and cervical cancer cells with hsp90 inhibitor induces proteasomal degradation and depletion of ATR and CHK1. This results in impairment of the DNA damage response (DDR) and causes sensitization to α-irradiation and replication stress due to hydroxyurea (Mol Cancer Ther 10:1194, 3011). In the present studies, we determined that treatment with pan-histone deacetylase (HDAC) inhibitor panobinostat (20 to 50 nM) or vorinostat (0.5 to 2 μM) induced polyubiquitylation and depletion of ATR and CHK1. Co-treatment with bortezomib, a proteasome inhibitor, restored PS-mediated depletion of ATR and CHK1. Notably, treatment with panobinostat induced hyperacetylation of both nuclear and cytoplasmic hsp90. This inhibited the chaperone association of nuclear hsp90 with ATR and CHK1, thereby promoting their degradation. PS treatment induced γ-H2AX levels, which were inhibited by co-treatment with N-acetylcysteine, suggesting that PS-induced ROS is involved in DNA damage. Moreover, PS treatment further increased α-irradiation induced DNA damage and its repair, characterized by the increase in α-irradiation-induced comet tail moment and the lack of DNA repair-mediated attenuation of the comet tail moment. To determine which class I HDAC is involved in deacetylation of nuclear hsp90, we individually knocked down (KD) HDAC 1, 2 and 3, by utilizing specific shRNA, and determined the effect on nuclear hsp90 hyperacetylation and the levels of ATR and CHK1. Our findings show that HDAC3 binds to hsp90 and KD of HDAC3, but not of HDAC1 or 2, caused hyperacetylation of nuclear hsp90 and depletion of ATR and CHK1. This was also observed in HDAC3 null mouse embryonic fibroblasts (MEFs). Ectopic overexpression of HDAC3 inhibited nuclear hsp90 acetylation in transformed cells. These findings demonstrate that HDAC3 is the nuclear hsp90 lysine deacetylase. They also demonstrate that genetic KD of HDAC3, or its inhibition by pan-HDAC inhibitors, induces hyperacetylation of hsp90, mediates loss of hsp90 chaperone association and depletion of ATR and CHK1, abrogates α-irradiation-induced DDR, and results in sensitization of transformed cells to DNA damage due to α-irradiation or replication stress.

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 4698. doi:1538-7445.AM2012-4698
Abstract 4700: A novel selective EZH2 inhibitor exhibits anti-tumor activity in lymphoma with EZH2 activating mutations
Authors: Creasy Caretha L. , McCabe Michael T. , Korenchuk Susan , Diaz Elsie , Ott Heidi , Thompson Christine S. , Ganji Gopi , Gorman Shelby A. , LaFrance Louis V. , Brandt Martin , McHugh Charles , Verma Sharad K. , Tummino Peter J.
Published: August 2012

EZH2 is the catalytic subunit of the Polycomb Repressive Complex 2 (PRC2) responsible for methylating histone H3 on lysine 27 (H3K27) and repressing transcription of target genes. Dysregulation of H3K27 methylation is implicated in tumorigenesis in both solid and hematopoietic tumors and occurs through multiple mechanisms including elevation of EZH2 due to loss of regulating miRNAs; inactivating mutations in UTX, an H3K27 demethylase which acts in opposition to EZH2; and somatic mutations in EZH2. Point mutations identified in EZH2 in both follicular lymphoma (FL) and GCB diffuse large B cell lymphoma (DLBCL) exhibit increased H3K27 tri-methylation due to an altered histone substrate preference. This suggests that inhibitors of EZH2 activity may be effective in treating lymphomas carrying activating mutations in EZH2. We have identified first-in-class inhibitors of EZH2 which are highly-potent, reversible, SAM competitive, selective and equipotent against both WT and mutant EZH2. Cellular mechanistic studies demonstrate that inhibition of EZH2 decreases global H3K27me3 in both WT and EZH2 mutant cell lines; however, the cell proliferation response is quite different. Overall, DLBCL cell lines harboring activating mutations in EZH2 are highly sensitive to EZH2 inhibition, while DLBCL cell lines with WT EZH2 are moderately sensitive or resistant. Transcriptional and ChIP-seq studies reveal differences in the transcriptional response of EZH2 target genes and methylation patterns among cell lines providing insight into the mechanism of sensitivity. In a mouse model bearing EZH2 mutant DLBCL tumor xenografts, inhibition of EZH2 methyltransferase activity reduces global H3K27me3, increases expression of EZH2 target genes and inhibits the growth of tumors in a dose dependent manner. Together, these data demonstrate for the first time that direct inhibition of EZH2 methyltransferase activity is effective at inhibiting tumor growth and suggests a promising path forward in the clinic for the treatment of EZH2 mutant lymphoma.

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 4700. doi:1538-7445.AM2012-4700
Abstract 4721: Adaptation to histone deacetylase inhibitors attenuates cancer cell growth
Authors: Lin Hsin-Ching , Liu Catherine , Aranake Amrita , Rabson Arnold B.
Published: August 2012

Histone deacetylase (HDAC) inhibitors, such as SAHA (suberoylanilude hydroxamic acid) inhibit the growth of multiple cancer cell lines in vitro, and human tumor xenografts in vivo. SAHA has recently been approved for the treatment of human T cell lymphomas and is in clinical trials for a number of other different malignancies. HDAC inhibitors lead to increased acetylation of multiple cellular proteins, including histones, and lead to major changes in cellular gene expression, however the precise mechanisms of their therapeutic effects are not known. HDAC inhibitors have been reported to have multiple effects on different cancer cell lines, ranging from induction of differentiation to induction of apoptosis, DNA damage, senescence, and autophagy. We hypothesized that chronic treatment of malignant cells with HDAC inhibitors, through changes in the program of cancer cell gene expression, would induce differentiation and senescence, and attenuate the malignant phenotype. We have treated a subclone of the SW480 colon cancer cells (S3 cells) with step-wise increasing amounts of SAHA. Chronic treatment with SAHA resulted in adaptation of S3 cells to SAHA, associated with marked decrease in cell growth and tumorigenicity in nude mice. In addition to slow growth, SAHA-adapted SW480 cells (designated SH80), exhibit features of differentiation including gland formation and increased intestinal alkaline phosphatase production (IAP), and increased cell size, associated with multiple nuclei and polyploidy. Interestingly, decreased growth rate and increased expression of alkaline phosphatase were seen even following drug withdrawal, suggesting that drug-adaptation resulted in a relatively stable phenotype. Frequent expression of senescence-associated β-galactosidase was observed in SH80 cells, particularly in giant cells and cell clusters, suggesting on-going senescence. Increased expression of p21 RNA and protein, previously suggested to contribute to the growth inhibition effect of HDAC inhibitors, was observed in the SH80 cells. SH80 cells also exhibited changes in the expression of several microRNAs previously implicated in oncogenesis, suggesting that altered expression of these miRs might contribute to the attenuated malignant phenotype in SH80 cells. These data suggest adaptation of colon cancer cells to HDAC inhibitor therapy may attenuate key aspects of the malignant phenotype, and offer a new therapeutic approach to the long-term control of 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 4721. doi:1538-7445.AM2012-4721
Abstract 4730: Inhibition of the p53-HDMX interaction sensitizes melanoma to chemotherapy
Authors: Russell Elisabeth A. , Gembarska Agnieszka , Marine Jean-Christophe , Bernal Federico
Published: August 2012

Melanoma is the leading cause of death from skin disease worldwide. It is an aggressive cancer that is often resistant to conventional chemotherapy and radiation-based therapy. The combination of a low survival rate in metastatic disease and lack of successful treatment options underscores the need to elucidate novel methods to target melanoma. More than 95% of human melanomas retain wild type p53, the protein responsible for maintaining genomic integrity; and HDMX, a negative regulator of p53 that neutralizes p53, has recently been found to be overexpressed in 65% of human cutaneous melanoma. These features suggest that targeting the p53-HDMX interaction in melanoma could not only restore the tumor suppressor function of p53, but also sensitize melanoma cells to treatment with conventional chemotherapeutic agents which rely on an intact p53 response. We have previously demonstrated that a hydrocarbon-stapled α-helix based on the transactivation domain of p53 (SAH-p53-8) efficiently disrupts p53-HDM2 and p53-HDMX complexes allowing for p53 to induce apoptotic cell death. Here, we show that SAH-p53-8 treatment is cytotoxic to metastatic melanoma cells by a p53-dependent mechanism. Additionally, SAH-p53-8 sensitizes melanoma to treatment with either cisplatin or the HDM2-specific inhibitor Nutlin-3. Importantly, the inhibition of the p53-HDMX complex arrests the growth of human metastatic melanoma in vivo. The restoration of functional p53 activity provides a basis for a previously unrecognized targeted therapy for metastatic melanoma.

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 4730. doi:1538-7445.AM2012-4730
Abstract 4733: Function of cytoplasmic histone deacetylase 5 is required for HIF-1α stability
Authors: Chen Shuyang , Ying Chengqian , Liang Dongming , Jethava Niti , Sang Nianli
Published: August 2012

Hypoxia is frequently associated with solid tumors and ischemic disorders, which are the top two leading causes of mortality in the world. Hypoxia-inducible factor 1 is a major regulator of angiogenesis and cellular adaptation to hypoxia. Accordingly HIF function has been considered a therapeutic target for multiple diseases. Histone deacetylase (HDAC) activity has been involved in regulating HIF function and HDAC inhibitors (HDACIs) have been documented to repress HIF function. However, histone deacetylases encompass multiple members, each has different cellular functions. HDACIs in clinical application or under development generally inhibit more than one HDAC. It is unclear which specific member of the HDAC family is involved in the regulation of HIF function. We present data here to show that histone deacetylase 5 (HDAC5) is required for HIF-1α protein stability. Knock-down of HDAC5 impaired hypoxic accumulation of HIF-1α and its function. In contrast, knock-down of HDAC1, HDAC3 or HDAC6 showed little or no effect. On the other hand, overexpression of HDAC5 enhanced HIF-1α stability. Specifically, cytosol localized HDAC5 mutant shows stronger effects on stabilizing HIF-1α compared to wild-type HDAC5. Mutation demolishing the deacetylase activity abolished the ability of HDAC5 to stabilize HIF-1α. Taken together, our data suggest that a cytosolic deacetylase activity of HDAC5 is required to stabilize HIF-1α. These findings also suggest that HDAC5 could be biological target for therapies based on modulating HIF function. This work is supported in part by grant R01-CA129494 (N.S.) from NCI, NIH.

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 4733. doi:1538-7445.AM2012-4733
Abstract 4734: Genes cooperatively downregulated by combined mTOR/histone deactylase (HDAC) inhibition are overexpressed in myeloma patients with lower survival
Authors: Michalowski Aleksandra M. , Simmons John K. , Patel Jyoti , Kuehl W Michael , Zhang Shuling , Landgren Ola , Mock Beverly A.
Published: August 2012

The molecular pathogenesis of many cancer types, including multiple myeloma (MM) and mantle cell lymphoma (MCL), involves alterations in the PI3K/Akt/mTOR and cyclin/CDK/CDKI/Rb (Rb) pathways. Previously, we showed that the combination of an HDAC inhibitor (HDACi) with rapamycin synergistically inhibited proliferation in 88% of human MM cell lines tested, and effectively controlled tumor growth in preclinical studies. To gain an initial understanding of the molecular mechanism of the synergistic action of the drug combination, we used an unbiased systems-level approach to analyze our gene expression profile (GEP) data with weighted gene co-expression network analysis (WGCNA). This analysis delineated the contribution of HDACi and rapamycin, singly and in combination, to the overall gene expression change of the combination by considering not only measures of fold-change and significance testing, but also the degree of gene expression inter-connectedness. WGCNA identified five gene modules, each representing a particular gene expression effect of the combination. Each gene module was individually tested for functional and clinical enrichment using gene set enrichment analysis (GSEA) and survival analyses with Cox regression. Of particular interest, the module containing genes cooperatively affected by both compounds was highly enriched (p<0.001) for genes involved in cell cycle (especially mitotic processes), immune recognition, and DNA damage/repair, which we investigated further. Genes down-regulated by the drug combination were most significantly correlated with genes over-expressed in MM patients. Furthermore, analysis of the cooperative drug signature in publicly available patient GEP datasets with survival annotation found it predictive of increased survival (p<0.01), thus linking the drug combination-induced transcriptional changes to predictions for enhanced survival.

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 4734. doi:1538-7445.AM2012-4734