bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5665: Multiplexing markers of response to sorafenib/mapatumumab combination therapy in hepatocellular carcinoma circulating tumor cells
Authors: Dolloff Nathan G. , Zhou Lanlan , Peters Kristi , Navaraj Arunasalam , Allen Joshua , Das Avisnata , Dicker David , El-Deiry Wafik
Published: August 2012

Hepatocellular carcinoma (HCC) accounts for approximately 600,000 deaths per year worldwide. Effective therapeutic strategies for advanced stage patients are lacking, and the median survival rate for patients who are not eligible for surgical resection is a mere 6 months. Circulating tumor cells (CTCs) are detectable in the peripheral blood of cancer patients, are accessible through a minimally invasive procedure, and may provide a source of tumor tissue for analyzing molecular markers of therapeutic response that may help guide treatment protocols. To demonstrate this potential, we developed a system for imaging and quantifying multiplexed molecular markers of response to sorafenib/mapatumumab combination therapy in an HCC CTC model. The sorafenib/mapatumumab combination regimen is currently in phase 2 clinical trials for the treatment of HCC, and molecular markers that mediate the synergistic anti-tumor activity of this combination have been identified. These markers include phospho-p42/44 MAPK (p-ERK), Mcl-1, c-IAP2, and apoptotic markers such as cleaved caspase-3. We developed a technical approach using MultiSpectral Imaging (MSI) to quantitatively measure multiplexed fluorescent signals from primary antibodies specific to phospho-ERK, Mcl-1, c-IAP-2, and cleaved caspase-3 that had been conjugated to quantum dots (Qdots) with distinct fluorescence emission spectra. We first confirmed the ability to modulate marker expression and uncouple multiplexed Qdot signals by MSI in cultured HCC cells that had been treated with sorafenib or sorafenib/mapatumumab combinations. We then applied this detection system to HCC cells that were spiked into human blood and recovered using FDA-approved CTC isolation technology (CellSearch CTC Test by Veridex). Our data demonstrate the potential for studying molecular markers of therapeutic response in CTCs. Information gained from this technology may be used to guide therapeutic decisions or serve as pharmacokinetic/pharmacodynamic endpoints in experimental clinical protocols.

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 5665. doi:1538-7445.AM2012-5665
Abstract 5673: Mechanisms responsible for the antitumor effect of metronomic chemotherapy with cyclophosphamide (Cy) and doxorubicin (Dox) in murine tumor models of mammary adenocarcinoma
Authors: Mainetti Leandro E. , Rozados Viviana R. , Rico María J. , Roggero Eduardo , Scharovsky O. Graciela
Published: August 2012

Metronomic chemotherapy (MCT) refers to the chronic, equally spaced, delivery of low doses of different chemotherapeutic drugs, without extended interruptions. We have already demonstrated that the administration, as an intervention strategy, of metronomic CY + DOX in M-406 and M-234p mammary adenocarcinomas-bearing mice inhibited tumor growth, increasing the survival rate. Our present aim was to study the mechanism/s by which that therapeutic effect was achieved. CBi and Balb/c inbred mice were s.c. challenged on day 0 with M-406 and M-234p mammary tumors, respectively, and treated from day 9 (M-406) and 10 (M-234p) on with: I) no further treatment (controls); II) CY in the drinking water (20mg/kg body weight/day); III) Dox (0.5 mg/kg i.p.), three times/week; IV) Treated as II + III. Blood samples were taken on days 0 and 20 (M-406) or 30 (M-324p) to evaluate VEGF and IL-10 serum levels determined by ELISA. Tumors were excised on days 25 and 31 for M-406 and M-234p, respectively, fixed and paraffin embedded for detection of Ki67 molecule by immunohistochemistry and apoptosis by the TUNEL technique. In the M-234p model the VEGF serum concentration of control animals (group I) was, on day 31, significantly higher than that measured on day 0 (p<0,005), while on day 31, groups II, III y IV showed lower values than group I (p<0.005) in the same day, and similar than those on day 0. The IL-10 serum levels in group IV were significantly higher than those found in group I and III. The apoptosis level in tumor cells in group IV was significantly higher when compared to control group (p<0.05). Also, the staining for the proliferation marker Ki67 showed values significantly lower in the tumors of animals treated with CY + DOX than those in group I. A similar behavior was seen in the M-406 tumor model. In conclusion, opposing to the expected results, the animals with the better therapeutic outcome showed the highest IL-10 concentration, a result that could indicate a possible immunostimulatory effect of this cytokine; the antitumor effect of MCT with CY plus DOX in mammary tumor models would be achieved, at least in part, by the inhibition of both tumor angiogenesis and proliferation in addition to the stimulation of tumor cells apoptosis.

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 5673. doi:1538-7445.AM2012-5673
Abstract LB-237: In vitro and in vivo characterization of CPI-267203, a potent Inhibitor of bromodomain-containing proteins
Authors: Normant Emmanuel , Cummings Richard , Bellon Steven , Bailey Christopher , Albrecht Brian , Hewitt Michael , Pardo Eneida , Campbell Robert , Jayaram Hari , Poy Florence , Sandy Peter , Gangurde Pranoti
Published: August 2012

The transcription factor c-Myc regulates diverse biological processes including growth, proliferation, differentiation, quiescence, and senescence or apoptosis. c-Myc proto-oncogene overexpression has been implicated in the genesis of many human malignancies. Identification of compounds that selectively and potently inhibit the expression of c-Myc hence could be beneficial for the treatment of these diseases. We and others have recently shown that inhibiting the binding of bromodomain-containing proteins such as Brd4 to their acetylated histone substrates leads to the specific suppression of c-Myc transcription. We report here the characterization of CPI-267203, a potent, selective and competitive inhibitor of BET domain proteins (Brd4 IC50 = 26 nM and CBP IC50 = 6.0 uM). In vitro, this molecule inhibits IL-6 production (EC50 = 11 nM) induced by lipopolysaccharide (LPS) in acute monocytic leukemia cells (THP1) and c-Myc mRNA production (EC50 = 27 nM) in Burkitt lymphoma cells (Raji). c-Myc mRNA and protein levels decline as early as 4hrs after treatment with CPI-267203, leading to cell cycle arrest and apoptosis. In vivo, CPI-267203 has 84% oral bioavailability and a plasma half-life of 1.9 h. In a xenograft model of human acute myelogenous leukemia (MV4:11), CPI-267203 inhibits the production of c-Myc mRNA and protein, inhibits tumor growth, and triggers tumor apoptosis. Reduction of c-Myc protein levels can also be detected in the normal skin of treated animals, suggesting that the level of c-Myc in the skin might be a useful surrogate pharmacodynamic marker to aid clinical trials.

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 LB-237. doi:1538-7445.AM2012-LB-237
Abstract LB-43: Community health advisors: a model in community outreach and health education
Authors: Kyles Brenda M.
Published: August 2012

The Shelby County Health Department's goal is to become the healthiest and safest community in Tennessee. Our mission is to promote, protect and improve the health and environment of all Shelby County residents. We place emphasis on the following programs: environmental health, epidemiology, health education, maternal and child health, immunizations, STD, HIV and AIDs, breast and cervical cancer screening and treatment and Tuberculosis management. We are committed to closing the gaps in health status and access to care among our community's diverse populations and reducing the occurrence of preventable disease and premature death among all Shelby County residents.

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 LB-43. doi:1538-7445.AM2012-LB-43
Abstract LB-440: Low density array profiling of head and neck squamous cell carcinoma utilizing a 25-gene signature can reliably identify hypoxic tumors
Authors: Williamson Amanda , Betts Guy NJ , Patiar Shalini , Valentine Helen R. , Irlam Joely J. , Ramachandran Anassuya , Merve Ashirwad , Homer Jarrod J. , Moller-Levet Carla , Buffa Franscesca M. , Hall Gillian , Miller Crispin J. , Harris Adrian L. , West Catharine ML
Published: August 2012

Purpose: Administering hypoxia modifying therapy to patients with head and neck squamous cell carcinoma (HNSCC) improves outcomes in those with identified hypoxic tumors. As no method for measuring tumor hypoxia has demonstrated clinical utility, this study aimed to validate use of a gene expression signature with a TaqMan Low Density Array (TLDA) platform. Experimental design: Tumor samples (n=201) from 80 HNSCC patients were collected prospectively from two centers. Fifty-three patients received pimonidazole prior to surgery. Hypoxia scores (TLDA HS) were obtained by quantitative real-time PCR (qPCR) using a 25-gene signature and customized TLDA cards. Assay performance was assessed as coefficient of variation (CoV). Relationships with outcome were determined by log-rank analysis. Results: The assay was sensitive with linear reaction efficiencies across a 4log10 range of inputted cDNA (0.001-10 ng/µl). Intra- (CoV=0.5%) and inter- (CoV=0.1%) assay reproducibility were excellent. Intra-tumor heterogeneity was lower for TLDA HS (23.2%) than for pimonidazole (67.2%) or single gene measurements of CA9 (62.2%), VEGFA (45.0%) or HIG2 (39.4%). TLDA HS in HNSCC cell lines increased with decreasing pO2 and was reduced but not completely abrogated in HIF1A silenced cells. TLDA HS correlated with Affymetrix U133 Plus 2.0 microarray HS (p<0.01) and pimonidazole scores (p=0.021). High TLDA HS was associated with worse overall (p=0.037) and recurrence free (p=0.036) survival. Conclusions: Gene expression measurements of hypoxia using a 25-gene signature and TLDA cards are sensitive, reproducible and associated with lower intra-tumor heterogeneity than assaying individual genes or pimonidazole binding. The approach is suitable for application in clinical trials.

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 LB-440. doi:1538-7445.AM2012-LB-440
Abstract LB-442: The nuclear matrix anchor domain of the DNA replication factor Ciz1 is commonly disrupted in tumours and is a circulating biomarker for lung cancer
Authors: Higgins Gillian , Cook Heather , Roper Katherine , Munckley Jennifer , Watson Irene , Blackhall Fiona , Rom William , Pass Harvey , Ainscough Justin , Coverley Dawn A.
Published: August 2012

The DNA replication protein Ciz1 promotes initiation of mammalian DNA replication in cooperation with cyclin A-dependent kinase, most likely by delivering cyclin A to sites where cyclin E-dependent pre-replication complex assembly has taken place. Normally, Ciz1 is anchored within nuclear matrix-associated foci that co-localize with sites of DNA replication, but in the absence of anchor domain Ciz1 retains the ability to promote initiation of DNA replication in isolated nuclei so attachment to the nuclear matrix is not essential for function. Expression of DNA replication and nuclear matrix anchor domains of Ciz1 are uncoupled and uneven at the transcript level in a wide range of common solid tumours, including breast and lung. In cell-based assays, recombinant anchor domain protein interferes with attachment of endogenous Ciz1 to the nuclear matrix, revealing a dominant negative effect that also impacts on nuclear matrix-recruitment of key components of the pre-replication complex. This suggests that Ciz1 normally plays a role in localizing initiation of DNA replication to the nuclear matrix. These findings implicate spatially unconstrained DNA replication as a source of nuclear disorder in cancer cells. We identified a variant Ciz1 isoform with alterations in the nuclear matrix attachment domain and tumour-restricted expression. RNAi-mediated selective inhibition of variant Ciz1 expression is sufficient to restrain the growth of tumour cells that express it, identifying variant Ciz1 as a functionally relevant driver of cell proliferation. We also present evidence that this form of Ciz1 is expressed in 34/35 lung tumours but not adjacent tissue, giving rise to stable protein quantifiable in less than a microlitre of patient plasma by western blot. Using two independent sets, with 170 and 160 samples, variant Ciz1 correctly identified stage 1 lung cancer patients with clinically useful accuracy. For set 1, mean variant Ciz1 level (+SD) in individuals without diagnosed tumours established a threshold that correctly classified 94% of small cell lung cancers (SCLC) and non-SCLC patients. Within set 2, comparison of stage 1 non-SCLC with asymptomatic age-matched smokers correctly classified 85% of patients, while comparison with individuals with benign lung nodules correctly classified 83% of patients. The data show that this cancer-specific, single marker is capable of indentifying early stage lung cancer within at-risk groups, without resort to invasive procedures.

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 LB-442. doi:1538-7445.AM2012-LB-442
Abstract LB-45: Real people- real stories: survivors teaching students
Authors: Fornia Judith L.
Published: August 2012

The Ovarian Cancer Alliance of Oregon and SW Washington is a proud partner member of the Ovarian Cancer National Alliance (OCNA). We actively bring the OCNA's program Survivors Teaching Students: Saving Women's Lives to the local level. Since 2002, the Ovarian Cancer National Alliance has been working to save the lives of women by helping healthcare practitioners learn to diagnose ovarian cancer earlier. Through OCNA's innovative educational program future healthcare professionals-physicians, nurse practitioners, nurses and physician assistants increase their understanding of ovarian cancer symptoms and risk factors so that they will be able to diagnose the disease when it is in its earlier, most treatable stages. Survivors Teaching Students brings ovarian cancer survivors into health professionals’ classrooms to share their stories and key information about the disease. At the local level, we also speak to pharmacy and naturopath students. Based on students’ evaluations we are accomplishing our goal of increasing awareness of this deadly cancer. Our local organization's mission is to promote awareness of ovarian cancer and to provide education to women and the health care community; to offer support to all ovarian cancer survivors by providing opportunities for networking, education and community outreach; and to advocate for public policy and funding for ovarian cancer research. In addition to the Survivors Teaching Students program, we are active in community health fairs/events, and have a presence in local Gynecological Cancer Support Groups.

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 LB-45. doi:1538-7445.AM2012-LB-45
Abstract LB-451: Treatment with hormonal blockade in estrogen receptor-positive tumors and correlation with molecular markers: a phase I pilot study of anastrozole in combination with everolimus, bevacizumab, sorafenib or erlotinib
Authors: Wheler Jennifer J. , Moulder Stacy , Falchook Gerald , Janku Filip , Naing Aung , Fu Siqing , Piha-Paul Sarina , Tsimberidou Apostolia Maria , Kurzrock Razelle
Published: August 2012

Background: Estrogen receptors are expressed across a variety of solid tumor types and may predict response to hormonal therapy. Objectives: Objectives were to evaluate safety, response signals and correlation with molecular aberrations. Methods: Beginning December 7, 2010 patients with estrogen receptor positive (ER+), advanced cancers were treated with anastrozole combinations. Toxicities were monitored and imaging evaluations were performed every 3 cycles (1 cycle = 4 weeks). Results: Patients were treated with anastrozole (1 mg) alone or in combination with everolimus (5 or 10 mg PO daily) (N = 18), bevacizumab (10mg/kg or 15 mg/kg IV q21d) (N = 5), sorafenib (200 mg PO BID or 400 mg qam/200 mg qhs) (N = 23), or erlotinib (75 mg PO daily) (N = 3). No dose-limiting toxicities or serious toxicities (≥ grade 3) were observed. On anastrozole/everolimus, 6/18 patients (33%) had either stable disease (SD) ≥ 6 months or partial response (PR) (breast cancer, 4; ovarian cancer, 2). 3 of 4 patients with SD ≥ 6 months or PR and available molecular results showed either PTEN loss or PIK3CA mutation or both. On anastrazole/bevacizumab, 1/5 patients (20%) had SD (11 months+; ovarian cancer). On anastrozole/sorafenib, 23/23 patients had progression of disease (PD). On anastrozole/erlotinib, 3/3 patients had PD. Conclusions: Treatment with anastrozole in combination with everolimus, bevacizumab, sorafenib or erlotinib is feasible and anastrozole/everolimus has demonstrated response signals in ER+ tumors (breast and ovarian), particularly in patients with PTEN loss or PIK3CA mutations. Study expansion and molecular profiling are ongoing.

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 LB-451. doi:1538-7445.AM2012-LB-451
Abstract LB-459: An essay concerning the molecular prediction of disease outcome in melanoma patients
Authors: Gschaider Melanie , Neumann Friederike , Peters Bettina , Lenz Florian , Cibena Micheal , Goiser Malgorzata , Wolf Ingrid , Wenzel Jörg , Mauch Cornelia , Schreiner Wolfgang , Wagner Stephan N.
Published: August 2012

Melanoma is the most lethal form of skin cancer. Current prognostic clinical and histopathological parameters are insufficient to accurately predict the outcome of individual patients. Several studies have claimed gene expression signatures to predict survival or metastasis of primary melanoma patients. However, the reproducibility among these studies is disappointingly low and led to the development of guidelines on statistical analysis and reporting of gene expression data for cancer outcome. We carefully followed these guidelines when we used gene expression data from primary human melanoma samples to identify gene signatures predictive for disease outcome. The class comparison dataset was established using microarray data from non-metastatic and metastatic primary melanoma samples. The top 50 differentially expressed genes were identified by Significance Analysis of Microarrays and used in an independent gene expression data set of primary melanomas for prediction. Multivariate Cox regression analysis determined the classifier revealing the best predictive value. This classifier comprised the Duffy blood group chemokine receptor and the ribosomal protein S27-like and defined the cutoff positions for favorable and detrimental disease outcome, respectively. We report this classifier as an essay to provide molecular information supplementary to standard morphological prognostic factors.

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 LB-459. doi:1538-7445.AM2012-LB-459
Abstract LB-468: miR-345 downregulation is associated with apoptosis resistance in pancreatic cancer: therapeutic implications
Authors: Srivastava Sanjeev K. , Bhardwaj Arun , Arora Sumit , Singh Seema , McClellan Steven , Wang Bin , Grizzle William E. , Singh Ajay P.
Published: August 2012

Resistance to apoptosis is one of the mechanisms underlying the highly metastatic and drug-resistant nature of pancreatic malignancy, which currently stands as the fourth leading cause of cancer-related death. Recent studies have identified several differentially-expressed microRNAs in pancreatic cancer, including miR-345, which is among the top aberrantly-expressed genes exhibiting significant downregulation. In this study, we have investigated the functional consequences of miR-345 downregulation in pancreatic cancer. We observed that miR-345 was significantly downregulated in our set of pancreatic cancer tissues as compared to the normal pancreas. Furthermore, a relative decrease in miR-345 levels was observed in poorly-differentiated pancreatic cancer cell lines as compared to the well-differentiated cell lines. Ectopic expression of miR-345 in two pancreatic cancer cell lines, MiaPaCa and Panc1, led to reduced cell viability and induction of apoptosis. miR-345 overexpressing pancreatic cancer cells exhibited loss of mitochondrial membrane potential, release of cytochrome c, activation of caspases 3/7 and PARP1 cleavage. In addition, we also observed translocation of apoptosis inducing factor (AIF) from mitochondria to nucleus and increased DNA fragmentation in miR-345 overexpressing pancreatic cancer cells. All these effects were reversed upon treatment of miR-345 overexpressing pancreatic cancer cells with anti-miR-345 oligonucleotides, thus confirming the role of miR-345 in apoptosis induction. Based on in silico analysis, we identified BCL2 as a candidate miR-345 target gene and experimentally validated the direct targeting of the 3′ UTR of BCL2 using a luciferase reporter assay. Consistent with this, miR-345 overexpressing pancreatic cancer cells exhibited a downregulated expression of BCL2 at the protein level. When we allowed unopposed exogenous expression of BCL2, it rescued pancreatic cancer cells from miR-345-induced apoptosis. Altogether, our data suggest that miR-345 downregulation is associated with gain of apoptosis resistance, and restoring its expression may have therapeutic effects in pancreatic 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 LB-468. doi:1538-7445.AM2012-LB-468
Abstract LB-471: Upregulation of miR-196a and HOTAIR is associated with the malignancy of gastrointestinal stromal tumors
Authors: Suzuki Hiromu , Nojima Masanori , Maruyama Reo , Yamamoto Eiichiro , Imai Kohzoh , Shinomura Yasuhisa
Published: August 2012

Although dysregulation of microRNA (miRNA) expression is a common feature of human malignancies, its involvement in gastrointestinal stromal tumors (GISTs) is not fully understood. In the present study, we aimed to identify miRNAs associated with the malignant potential of GISTs. miRNA microarray analysis enabled us to detect the frequent upregulation of miR-196a in high-risk GISTs. Quantitative RT-PCR analysis of 56 fresh frozen and 100 formalin-fixed paraffin-embedded (FFPE) specimens confirmed that overexpression of miR-196a is associated with a high-risk grade, metastasis and poor survival among GIST patients (hazard ratio, 9.1; P < 0.001). The miR-196a genes are located within the HOX gene clusters, and gene expression microarray analysis revealed that HOXC genes and a large intergenic non-coding RNA (lincRNA), HOTAIR, were concurrently upregulated in GISTs overexpressing miR-196a. Elevated HOTAIR expression was also strongly associated with the high-risk category, metastasis and poor clinical outcome of GISTs (hazard ratio, 9.0; P = 0.034). RNAi-mediated knockdown of HOTAIR suppressed the invasiveness of GIST cells, and induced a significant change in the expression profile of genes that included previously reported HOTAIR target genes. This is the first report of concurrent overexpression of HOX genes and collinear non-coding RNAs in human malignancy, and our data suggest that miR-196a and HOTAIR could be useful biomarkers and novel therapeutic targets in malignant GISTs.

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 LB-471. doi:1538-7445.AM2012-LB-471
Abstract LB-472: p63 inhibits epithelial - mesenchymal transition by promoting the expression of miR205 in human bladder cancer cells
Authors: Tran Mai N. , Choi Woonyoung , Navai Neema , Wszolek Matthew F. , Lee I-ling C. , Siefker-Radtke Arlene O. , McConkey David J.
Published: August 2012

Epithelial - mesenchymal transition (EMT) is a physiological process in normal tissue development and wound healing that has been shown to play important roles in tumor metastasis, “stemness,” and drug resistance. Therefore, it is critical to understand the molecular mechanisms that control EMT. EMT is typically characterized by the loss of the epithelial marker E-cadherin and gain of the transcriptional repressors of E-cadherin (Zeb1, Zeb2, Twist, SnaiI, and Slug). MicroRNAs belonging to the mir200 family and miR205 are involved in EMT through suppression of Zeb1 and Zeb2. Recently, p53 has been demonstrated to regulate EMT by transcriptionally activating the microRNA mir-200c, which in turn suppresses Zeb1; however, the mechanisms controlling miR205 remain elusive. We noticed that expression of the p53 homologue p63 correlates with the expression of E-cadherin in bladder cancer cell lines, suggesting that p63 might participate in regulating EMT. Consistent with this idea, expression of p63 and miR205 are strongly correlated with each other in a large panel of bladder cancer cell lines. Interestingly, stably knocking down (KD) p63 in two epithelial cell lines (UM-UC6 and UM-UC14) repressed the expression of miR205. Consistent with the role of miR205 in suppressing Zeb1, p63 knockdown resulted in increased expression of Zeb1 in the UM-UC6 cells, and this effect was reversed by expression of exogenous miR205. p63 knockdown reduced both the primary and mature forms of miR205, suggesting that p63 might transcriptionally activate miR205 gene expression. Intriguingly, chromatin immunoprecipitation (ChIP) revealed that p63 binds to a putative regulatory region of miR205 in UM-UC6 and UM-UC14. Furthermore, the acetylated histone H3 mark (indicative of active/open chromatin) was enriched in the region encompassing the p63 binding site. Together, our data identifies p63 as an important repressor of EMT via its ability to promote expression of miR205, which represses Zeb1 expression.

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 LB-472. doi:1538-7445.AM2012-LB-472
Abstract LB-474: Estrogen receptor regulated long non coding RNA: An evolving paradigm in prostate cancer progression
Authors: Chakravarty Dimple , Sboner Andrea , Giannopoulou Eugenia , Elemento Olivier , Fox Archa , Rubin Mark
Published: August 2012

Prostate cancer is the second leading cause of cancer death in men. Anti-androgen therapy has been the preferred modality of treatment for several years, however prostate tumor co-opts alternate molecular mechanisms to overcome need for androgen and thereby become “castrate resistant”. Several prognostic markers have received functional evaluation in clinical setting; unfortunately current biomarkers are not accurate enough to predict disease progression and/or response to anti-androgen therapy. Thus there is an unmet need for novel biomarkers. Several studies have indicated that Estrogen receptor (ER) plays an important role in different types of tumors and while the importance of ER/E2 signaling pathway has been extensively investigated in breast cancer and other gynecological malignancies, the impact of this pathway on malignancies associated with other tissues like prostate warrants further investigation. Here we report a novel ERα- lncRNA signaling axis in prostate cancer progression. Our data provides compelling evidence to demonstrate that expression of ERα can “over-ride” requirement of androgens or androgen receptor during prostate cancer progression. Using a combination of ChIP-seq and RNA-seq approach we successfully identified a compendium of ER regulated lncRNA. We have utilized several biochemical assays and invivo xenograft studies to demonstrate the oncogenic relevance of ERα regulated lncRNA. Expression of lncRNA correlated well with clinicopathological variables in several patient cohorts. Finally we have tested therapeutic efficacy of targeting these oncogenic lncRNA using siRNA encapsulated chitosan based nanoparticles either single or in combination with current treatment modalities. In conclusion we have identified ER-regulated lncRNA as novel biomarkers for prostate cancer progression and therapy resistance in patients with advanced PCa.

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 LB-474. doi:1538-7445.AM2012-LB-474
Abstract LB-480: MicroRNA in situ hybridization techniques: Co-detection of target protein and image analysis-based quantitation
Authors: Nielsen Boye S. , Moller Trine , Holmstrom Kim
Published: August 2012

MicroRNAs (miRNAs) are small noncoding RNAs that constitute a novel group of biomarkers with exciting functions in cell differentiation, proliferation and apoptosis by mediating degradation or destabilization of target mRNAs. Expression profiling provides identification of disease-related miRNAs; however, questions towards molecular interactions in vivo remain un-answered. In situ hybridization (ISH) allows visualization the miRNAs in histological sections and thereby allows determination of specific cellular origin, which is an essential step for understanding the miRNAs role in any biological setting. We have developed fast double fluorescence and quantitative ISH technologies that facilitates interpretation of roles for miRNA in cancer progression. On a semi-automated platform, HistoFlex, the double fluorescence assay can be completed within 2 hours without loss of sensitivity and specificity. In order to characterize the miR-21 positive cells in the breast cancer stroma, we combined miR-21 ISH with smooth muscle α-actin in a double fluorescence assay, and found that most, but not all miR-21 positive cells, are smooth muscle α-actin positive and include both myofibroblasts and myoepithelial cells in the neoplastic tissue. Subsequent combination of miR-21 ISH and programmed cell death-4 (PDCD4) immunofluorescence showed differential expression in many but not all miR-21 positive cells. The findings suggest that PDCD4 (mRNA), as a predicted miR-21 target according to sequence alignment, may also take place in vivo. Thus, a role of miR-21 in cell survival and apoptosis is likely. miRNA ISH also allows quantitative estimation of miRNA expression levels in archived formalin-fixed and paraffin embedded tissue samples. Using chromogenic detection of miRNA and subsequent image analysis, semi-quantitative expression estimates can be obtained, which can be used for correlation with clinical parameters, such as drug response, diagnosis, prognosis and other molecular profiles. We have established an automated 1-day assay that provides quantitative estimates with a precision of app. 15%. We also show that a quantitative estimate obtained in one paraffin section with tumor tissue is representative for the whole tumor. For clinical applications, we present data showing that the miRNA ISH signal can be quantified in routinely processed paraffin samples from colon and breast cancer, and that this approach can provide novel diagnostic possibilities.

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 LB-480. doi:1538-7445.AM2012-LB-480
Abstract LB-483: p53-dependent release of Alarmin HMGB1 is a central mediator of senescent phenotypes
Authors: Davalos Albert R. , Kawahara Misako , Malhotra Gautam , Beausejour Christian , Rodier Francis , Campisi Judith
Published: August 2012

Cellular senescence irreversibly arrests the proliferation of cells at risk for malignant transformation in part through activities of the transcriptional regulator and tumor suppressor p53. Cells that senesce owing to DNA damage also secrete many biologically active factors, including inflammatory cytokines such as IL-6. The High Mobility Group Box 1 (HMGB1) protein is unusual in having two distinct functions. Intracellularly, it binds chromatin and modulates transcription, including stimulating p53 activity. In addition, necrosis or microbial infection causes HMGB1 leakage or active secretion, respectively, whereupon it functions as an extracellular Alarmin to signal tissue damage and promote tissue regeneration, stem cell recruitment and immune activation. We show that HMGB1 is largely nuclear in non-senescent human and mouse fibroblasts and epithelial cells, but is actively exported from the nucleus and secreted by senescent cells. In culture and in vivo, HMGB1 re-localization occurred prior to the appearance of other hallmarks of senescence, and depended on the function of p53, but not the upstream p53 activator ATM. Old mice contained significantly higher levels of HMGB1 in serum compared to young animals. Disruption of HMGB1 stoichiometry, either by overexpression or depletion, induced a p53-dependent senescence growth arrest, but only HMGB1 overexpression, not HMGB1 depletion, promoted IL-6 secretion. Senescence-associated secretion required endogenous and secreted HMGB1 because deletion of endogenous HMGB1 – or addition of an HMGB1 blocking antibody - attenuated IL-6 secretion. Recombinant HMGB1 protein induced IL-6 secretion in cells depleted, but not harboring, endogenous HMGB1. Depletion of endogenous HMGB1 promoted NF-κ B transcriptional activity in cells cultured with recombinant HMGB1. Our findings identify a novel biological setting (senescence), independent of necrosis or microbial infection, in which HMGB1 secretion occurs, and link senescence-dependent HMGB1 redistribution to p53 activity and inflammatory cytokine secretion.

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 LB-483. doi:1538-7445.AM2012-LB-483
Abstract LB-486: Overexpression of a mutated, constitutively active HIF2α promotes the carcinogenesis of TRACK mouse
Authors: Fu Leiping , Wang Gang , Shevchuk Maria M. , Nanus David M. , Gudas Lorraine J.
Published: August 2012

Background: VHL disease patients have an increased risk of developing clear cell renal cell carcinomas (ccRCC). Increased expression of HIF1α and/or HIF2α has been proposed as a key step in ccRCC carcinogenesis. We recently reported a mouse model of VHL renal cancer, the TRACK model (Fu et al, Cancer research. 2011;71(21):6848-56.). In the TRACK model, proximal tubule (PT) cell overexpression of a triple mutant (P402A, P564A and N803A) HIF1α in the mouse mimics human VHL kidney disease. Methods: Here we report a mouse model expressing a triple mutant (P405A, P531A and N847A) HIF2α in PT cells. Mutant HIF2α was expressed in both WT and HIF1α-expressing C57BL/6 mice. Results: Transgenic (TG) mice expressing this HIF2α construct specifically in PT cells exhibit vacuolization consistent with glycogen accumulation and hydropic degeneration. The activation of HIF2α was confirmed by increased expression of HIF2α target genes, e.g. CA9. Neoplastic transformation was not observed in the kidneys of HIF2α TG+ mice. In HIF1α/HIF2α-expressing mice, disorganized PT cells mimicking in situ carcinoma was observed in 5 mo old mouse kidneys, which was seen only at 14 mo in mice expressing only mutant HIF1α. Conclusions: These data suggest HIF2α accelerates carcinogenesis in the mutant HIF1α TRACK model. These mouse models provide excellent models to study VHL renal disease and carcinogenesis, and provide an opportunity for chemoprevention studies.

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 LB-486. doi:1538-7445.AM2012-LB-486
Abstract LB-495: Myeloid-derived suppressor cells promote cancer-associated fibroblast migration and carcinoma cell invasion
Authors: Shaw Aubie K. , Novitskiy Sergey , Moses Harold L.
Published: August 2012

Metastasis the most devastating part of cancer, yet we know very little about the earliest steps of metastasis. Myeloid-Derived Suppressor Cells (MDSC) are CD11b+, Gr-1+ bone marrow-derived cells that have been shown to promote tumor development by suppressing T cells. In addition to immune suppression, MDSC can promote metastasis. MDSC localize to the invasive edges of primary tumors and promote breast cancer lung metastasis. Localized invasion of cancer cells is the earliest step of metastasis and fibroblasts are required for breast cancer cell invasion. Fibroblasts remodel the ECM and leave tracks for collective migration of carcinoma cells. These studies seek to identify the mechanism of MDSC promotion of metastasis by determining if MDSC can stimulate fibroblast migration and lead localized carcinoma invasion. Freshly isolated MDSC from a mammary tumor-bearing mouse increase fibroblast migration. TGFβRII-/- fibroblasts demonstrate a further increase in migration in response to MDSC, suggesting that TGFβ signaling is not required for MDSC promotion of fibroblast migration. MDSC secretions act directly on fibroblasts since conditioned medium from MDSC increases fibroblast migration to the same extent as live MDSC. Antibody array analysis identified several MDSC secreted proteins, including CXCL11, CXCL15, FGF2, IGF-I, IL-4 and Shh. MDSC increase carcinoma cell invasion through Matrigel, but only when fibroblasts are present. MDSC treatment of fibroblasts stimulates Akt and ERK1/2 phosphorylation and loss of secreted proteins, including SDF-1, TIMP-1, TNFα and VEGF. Analysis of subsets of MDSC identified that monocytic MDSC (CD11b+, Ly6Chi, Ly6G-) increase fibroblast migration more than other myeloid cell populations. Tumor-derived MDSC promote fibroblast migration more that splenic MDSC. These studies show that MDSC can lead fibroblasts to stimulate carcinoma invasion and suggest that perivascular MDSC may be the earliest stimulus for localized invasion during metastasis.

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 LB-495. doi:1538-7445.AM2012-LB-495
Abstract LB-501: Role of HGF in epithelial-stromal cell interactions during progression from benign breast disease to ductal carcinoma in situ
Authors: Casbas-Hernandez Patricia , Troester Melissa A. , Perez Erick Roman , Sandhu Rupninder , Kirk Erin , D'arcy Monica , Rein Jessica
Published: August 2012

Role of HGF in epithelial-stromal cell interactions during progression from benign breast disease to ductal carcinoma in situ. Introduction: Tumor-stroma interactions create a ‘permissive microenvironment’ during cancer progression. Basal and luminal breast cancers (BC) have distinct tumor-stroma interactions, but few studies show how gene expression evolves in premalignant cells. We observed that hepatic fibrosis/HGF signaling is induced in basal-like microenvironments, and previous studies have demonstrated that overexpression of HGF is a predictor of breast cancer recurrence and survival. However it is not known whether HGF signaling is altered in early (premalignant and DCIS) lesions. Methods: To study tumor-stroma interactions during cancer progression, we cocultured breast fibroblasts with a panel of isogenic cell lines, the MCF10 series. The MCF10 series represents different stages of cancer progression (benign, atypical hyperplasia and ductal carcinoma in situ). RNA from cocultures was used to perform gene expression microarrays and proteins secreted into media were analyzed by antibody arrays. Differentially expressed genes were identified using supervised analysis, followed by gene ontology analysis. In addition, a signature of basal-like microenvironment was evaluated to test whether the stroma-epithelial interactions observed in basal-like cancers were also present in premalignant cells. Finally, HGF signaling was inhibited (using an HGF-antibody) to assess effects of this pathway on migration and gene expression. Results: Cocultures with MCF10DCIS express higher levels of RNA involved in immune response processes and connective tissue disorders, as reported previously for basal-like BC. In contrast, premalignant MCF10AT and MCF10A cells showed weak expression of these profiles. In cytokine arrays, striking overexpression of HGF was observed in both MCF10AT1 and MCF10DCIS cocultures relative to cocultures with the benign MCF10A cell line, with corresponding increases in c-MET levels. These changes correlated with increased migration, which was blocked by adding HGF-specific antibodies to the media. Conclusions: Inhibition of HGF alters stromal-epithelial interactions, rendering the premalignant microenvironment more similar to normal microenvironments. HGF signaling is necessary for stroma-induced migratory phenotypes in vitro and is induces widespread gene expression changes. Targeting HGF signaling in early carcinogenesis may have value in preventing progression of basal-like precursor lesions.

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 LB-501. doi:1538-7445.AM2012-LB-501
Abstract LB-506: Targeted integrations of fluorescent proteins into STAT3 and STAT1 Loci via ZFN mediated homologous recombination in human cancer cell lines provides a suitable and adequate model system for cell-based testing of new candidate drugs for human tumor treatment
Authors: Samsonov Andrey , Zhang Fan , Zhang Hongyi , Zenser Nathan , Fetter John , Malkov Dmitry
Published: August 2012

STAT3 which is a member of the family of signal transducers and activators of transcription is an oncogene and it is constitutively activated in numerous cancer cell lines and human cancers including breast, lung, head and neck, liver, and pancreas. Another STAT family member STAT1 possesses cancer-inhibitory properties and once activated may promote apoptosis in tumor cells. Thus STAT3 has been identified as a promising drug discovery target for many cancers. Ideally the potential inhibitors of STAT3 should be very selective and should not inactivate STAT1. To better address these cancer-research important genes, we tagged STAT3 and STAT1 loci with fluorescent protein (FP) sequences (RFP and GFP respectively) by targeted integration via Zinc-Finger Nuclease (ZFN) mediated homologous recombination in cancer derived cell lines. ZFN technology is a fast and reliable way to manipulate the genome in a targeted fashion. ZFNs are synthetic proteins engineered to bind DNA at a sequence-specific location and create a double strand break (www.compozrzfn.com). The cell's natural machinery repairs the break in one of two ways: non-homologous end joining or homologous recombination. Utilizing the homologous recombination pathway that ZFNs induce, we successfully inserted the FP transgenes into the desired target locations - at the N-terminus of the STAT3 locus and at the C-terminus of the STAT1 locus. Three cell lines were created, two with the single STAT tagged and a third with both STATs tagged. Integration resulted in endogenous expression of fluorescent STAT3 and STAT1 chimeric fusion proteins in the A549 cell lines. Functional analyses of knock-in cell lines indicated that both STAT3 and STAT1 native gene regulation is conserved resulting in normal levels of protein expression and preservation of protein function. When stimulated with IL-6 or INF-γ, A549 cells showed fast and robust nuclear translocation of RFP-STAT3 or STAT1-GFP respectively. Cell pre-incubation with a known specific STAT3 inhibitors showed that STAT1-GFP, INF-γ-induced translocation was not impaired. Thus, these cell lines provide a simple and convenient method for high-content screening and pre-clinical testing of potential STAT3 inhibitors while ensuring that the STAT1 pathway is not affected. This approach of zfn-tagging endogenous genes could be applied to other cancer targets.

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 LB-506. doi:1538-7445.AM2012-LB-506