bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4011: Why does this APC exon 9 mutation causes severe and not attenuated familial adenomatous polyposis (FAP)
Authors: Cheah Peh Yean , Wong Yu Hui , Loi Carol , Koh Poh Koon , Tang Choong Leong
Published: August 2012

Familial adenomatous polyposis (FAP), an autosomal dominantly inherited form of colorectal cancer (CRC), is characterized by up to thousands of adenomatous polyps in the colon and rectum of an affected individual by the second or third decade of life. The risk of cancer is virtually 100% if the polyps are not removed in time. Germline mutation in the tumor suppressor, adenomatous polyposis coli (APC) gene causes the majority (80%) of FAP. Point mutation in 5′end of exon 9 of APC usually results in an attenuated form of FAP (aFAP), characterized by later age of onset (forth or fifth decade of life) and much less (<100) polyps. The presence of a physiological in-frame isoform with exon 8 spliced to 3′end of exon 9 (exon 9a) dilutes any deleterious effect of the mutation. We report here an interesting case of a proband with an APC mutation in 5′end of exon 9 that presented with 6 synchronous advanced CRC at age 37. The novel insertion-deletion (indel) mutation at codon 409 removes an exonic splice enhancer site that causes skipping of the whole of exon 9 in the mature transcript, resulting in a premature stop codon at exon 10 and a truncated protein that removes all of the β-catenin (CTNNB1) binding motifs, thus activating the downstream T-cell transcription factor (Tcf) pathway. This surprising consequence of the indel would not have been identified if the mutation screening has not begun with the protein truncation test and the APC transcript. The finding indicates the necessity of examining any point mutation in the 5′end of exon 9 for exonic splice enhancer or repressor site to avoid clinical mismanagement and counseling based on just the mutation site.

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 4011. doi:1538-7445.AM2012-4011
Abstract 4016: Epigenetic regulation of CXCR3 splicing in prostate cancer cells
Authors: Kumar Devi Sharanya Sampath , Wells Alan
Published: August 2012

Prostate cancer is the second leading cause of cancer death among men, with the metastasis of these tumors being the main reason for the high rates of mortality and morbidity. This tumor progression requires epithelial cell dedifferentiation, migration, invasion and growth in an ectopic site. CXCR3 is a G-protein coupled receptor whose splicing was found to be differentially regulated in prostate cancer. We have found over-expression of the CXCR3A splice isoform in prostate cancers and cell lines whereas normal prostate epithelium expresses by and large only the CXCR3B isoform, representing a possible switch in the expression between the CXCR3 splice variants. As these variants link differentially to pro- and anti-invasion signaling pathways, this may explain the relentless progression of the prostate carcinoma cells. The switch is present at both the RNA and the protein level which suggests that the altering factor is at the trancriptional control level. We hypothesized that a mutation in the gene or an epigenetic modification of the genome could possibly be regulating the splicing event and hence the switch in the expression. CXCR3 when scanned for mutations was found to be intact except for a polymorphism in the intronic region which coincided with the normal prostate cells. On the epigenetic regulation side, the CpG dinucleotides in the regulatory regions, the one intron and 5′ upstream region, susceptible to methylation changes were analysed for methylation differences. The upstream sequence does not show a differential methylation pattern whereas the intronic region shows differential methylation between the cancer and normal prostate cells at 3 different nucleotides. A possible explanation is that the methylation in the introns alters the speed of the transcription machinery allowing more time for a particular splice variant to be transcribed more than the other. A bichromatic reporter minigene has been designed for each of the differentially methylated CpG nucleotides, to study its effects on the splicing regulation of the CXCR3 gene. Further studies include expressing the minigene and documenting the splicing differences.

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 4016. doi:1538-7445.AM2012-4016
Abstract 4033: A20 gene promoter methylation confers resistance to colorectal carcinoma cell lines
Authors: Siraj Abdul K. , Al-Rasheed Maha , Bavi Prashant , Hussain Azhar , Al-Sanea Nasser , Al-Dayel Fouad , Uddin Shahab , Al-Kuraya Khawla S.
Published: August 2012

Gene silencing associated with aberrant methylation of promoter region CpG islands is an acquired epigenetic alteration. It serves as an alternative to genetic defects in the inactivation of tumor suppressor and other genes in human cancers. Several genes have been shown to be epigenetically inactivated in a wide range of tumors and most neoplasms show hypermethylation of one or more genes. A20, also known as TNF alpha induced protein3 (TNFAIP3), is a tumor suppressor gene and a well-known negative regulator of the NF-kB pathway. In colorectal carcinoma (CRC), aberrant NF-kB regulation has been associated with poor prognosis and resistance to therapy. Previously, we elucidated the biological role of A20, mechanism of its inactivation and its prognostic role in colorectal carcinoma. Loss of A20 protein expression by immunohistochemistry and A20 deletions by FISH were studied in a microarray cohort of CRC cases and cell lines for mutations in A20 gene and A20 promoter methylation were also screened. We further elucidated the mechanism of A20 inactivation in colorectal carcinoma cell lines in this extended study. Methylation status of A20 promoter in CRC cell lines correlated with the degree of A20 protein expression: unmethylated cell line SW948 showed the highest expression of A20 protein whereas LOVO, which was methylated, showed almost complete loss of A20 expression. CRC cell lines with high levels of A20 expression (SW948 and COLO320), intermediate levels (HCT-15 and DLD-1) and very low levels (LOVO-1) were immunoblotted for detection of A20 protein expression. Treatment of methylated colorectal cell lines with the methyltransferase inhibitor, 5-aza-2′deoxy-cytidine resulted in partial demethylation and re-expression of A20 gene, suggesting the role of methylation in silencing the gene. In addition, demethylation of A20 gene by 5-aza-2′deoxy-cytidine also decreased cell viability by 15-20X in CRC cell lines, suggesting that A20 expression may play an important role in CRC tumorigenesis. In conclusion, A20 methylation may be a promising molecular marker to predict patient outcome in colorectal cancer, and may be used as therapeutic demethylating target.

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 4033. doi:1538-7445.AM2012-4033
Abstract 404: Regulation of NKG2D expression and NKG2D-positive T cell tumor-specific trafficking by doxorubicin and IL-12 in vivo
Authors: Hu Jiemiao , Zhu Shiguo , Xia Xueqing , Yokoyama Wayne M. , Li Shulin
Published: August 2012

IL-12 and doxorubicin treatment are known to polarize anti-tumor immune response and induce tumor cell death, respectively, when each is administered alone. IL-12 treatment can boost NKG2D receptor expression on immune cells, enhancing NK cell cytotoxicity against different tumor cell lines. However, IL-12 treatment alone can only modestly increase expression of NKG2D receptor. In tumor immuno-surveillance, it is important to create an environment in which spleen cells constitutively express the NKG2D receptor thereby triggering NKG2D-dependent tumor cell death. In an earlier study, we found that coadministration of the anti-neoplastic drug doxorubicin and IL-12 greatly boosted anti-tumor efficacy. This increased anti-tumor efficacy was associated with a substantial increase in immune cell infiltration at tumor sites, but the underlying mechanism by which the increased number of immune cells was trafficked into the tumors remains largely unknown. This report demonstrates that doxorubicin plus IL-12 treatment induces the higher expression of NKG2D receptor in CD8 T cells than IL-12 treatment alone, resulting in an increased infiltration of NKG2D-positive CD8 T cells, and leading to an NKG2D-dependent tumor growth inhibition in vivo. These in vivo observations are in agreement with our in vitro result, in which the encounter between NKG2D receptor-positive effector cells and NKG2D ligand-positive tumor cells yields an NKG2D-dependent tumor cell death as determined by cytolytic activity assay. Blocking the NKG2D receptor by using neutralization antibody impaired doxorubicin plus IL-12 treatment-mediated tumor eradication in vivo and effector cell-mediated tumor cell death in vitro. Together, the evidence links the higher IL-12 plus doxorubicin treatment-induced NKG2D expression to greater CD8 T cell infiltration into tumors, tumor cell death, and anti-tumor efficacy.

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 404. doi:1538-7445.AM2012-404
Abstract 4044: Epigenetic repression of RARRES1 is mediated by methylation of a critical proximal promoter and a loss of CTCF binding
Authors: Lin Huey-Jen L. , Peng Zhengang , Shen Rulong , Li Yingwei , Teng Kun-Yu , Shapiro Charles
Published: August 2012

Background: The critical promoter element responsible for epigenetic silencing of retinoic acid receptor responder 1 (RARRES1) by methylation is unclear. Likewise, how aberrant methylation interplays effectors and thus affects breast neoplastic features remains largely unknown. Methodology/Principal Findings: We first compared methylation occurring at the sequences flanking the RARRES1 promoter in primary breast carcinomas to that in adjacent benign tissues. Surprisingly, tumor cores displayed significantly elevated methylation occurring solely at the upstream region, while the downstream element proximal to the transcription start site (+1) remained largely unchanged. Yet, hypermethylation at the former did not result in appreciable silencing effect. In contrast, the proximal sequence displayed full promoter activity and methylation of which remarkably silenced RARRES1 transcription. This phenomenon was recapitulated in breast cancer cell lines, in which methylation at the proximal region strikingly coincided with downregulation. We also discovered that CTCF occupancy was enriched at the unmethylayed promoter enriched with transcription-active histone markings. Furthermore, knocking-down CTCF expression hampered RARRES1 expression, suggesting CTCF positively regulated RARRES1 transcription presumably by binding to unmethylated promoter poised at transcription-ready state. Moreover, RARRES1 restoration not only impeded cell invasion but also promoted death induced by chemotherapeutic agents, denoting its tumor suppressive effects. Its role of attenuating invasion agreed with data generated from clinical specimens revealing that RARRES1 was generally downregulated in metastatic lymph nodes compared to the tumor cores. Conclusion/Significance: For the first time, this report delineated that silencing of RARRES1 by hypermethylation is occurring at a crucial proximal promoter element and is associated with a loss of binding to CTCF, an activator for RARRES1 expression. We also revealed the tumor suppressive roles exerted by RARRES1 in part by promoting breast epithelial cell death and by impeding cell invasion that is an important property for metastatic spread.

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 4044. doi:1538-7445.AM2012-4044
Abstract 4045: Intragenic ATM methylation in peripheral blood DNA as a biomarker of breast cancer risk
Authors: Brennan Kevin , Garcias-Closas Montserrat , Orr Nicholas , Fletcher Olivia , Jones Michael , Ashworth Alan , Swerdlow Anthony ,   KConFab Investigators, Riboli Elio , Vineis Paolo , Dorronsosro Mirren , Clavel Francoise , Panico Salvatore , Onland-Moret N. Charlotte , Trichopoulos Dimitrios , Kaaks Rudolf , Khaw Kay-Tee , Brown Robert , Flanagan James M.
Published: August 2012

DNA methylation is an important epigenetic modification that could affect cancer risk through direct effects on disease, and/or mediating effects of genetic and environmental factors. Global and gene-specific DNA methylation levels in white blood cells (WBC) have been related to risk of breast and other cancers, however, evidence is generally weak. We analysed WBC DNA methylation of two ATM intragenic loci (ATMmvp2a and ATMmvp2b) and genome-wide DNA methylation in LINE1 repetitive elements in three breast cancer studies with pre-diagnostic blood samples. Studies included a case-control study with cases derived from a prospective cohort of high-risk breast cancer families (KConFab), and nested case-control studies in two prospective cohorts: the Breakthrough Generations Study (BGS) and the European Prospective Investigation into Cancer and Nutrition (EPIC). Total sample size was 640 incident cases of invasive breast cancer and 741 controls. Bisulphite pyrosequencing was used to quantify methylation in WBC DNA. Quintile analyses of the combined data for ATMmvp2a showed an increased risk of breast cancer limited to women in the highest quintile of methylation (OR =1.89 (1.36-2.64), p= 1.64x10-4). We found no significant differences in estimates across studies, however, the largest differences in ATMmvp2a methylation levels were observed for familial cases in KConFab compared to controls. We observed no significant differences in LINE1 or ATMmvp2b methylation levels between cases and controls. These results indicate that WBC DNA methylation levels at ATM could be a marker of breast cancer risk and further support the pursuit of epigenome-wide association studies in peripheral blood DNA methylation. Funding: Breast Cancer Campaign Fellowship and Cancer Research UK Program C536/A6689.

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 4045. doi:1538-7445.AM2012-4045
Abstract 4047: Introducing a novel acoustic imaging tool for breast cancer detection
Authors: Alizad Azra , Whaley Dana H. , Urban Matthew M. , Greenleaf James F. , Fatemi Mostafa
Published: August 2012

Objective: This paper introduces an alternative non-invasive and non-ionizing breast imaging tool called vibro-acoustography (VA). This method is based on low-frequency harmonic vibrations induced in the object by the radiation force of ultrasound. Contrast in Vibro-acoustography images represent several properties of the object, including the dynamic characteristics at the audio frequency and scattering and absorption properties at the ultrasound frequency. X-ray mammography and ultrasound are the main imaging modality used today for detection of breast lesions. X-ray mammography, however, is ionizing and its sensitivity is greatly reduced in dense breasts. Ultrasound images suffer from speckles or other or artifact that prevents the physician from delineating the mass lesion from the background tissue. Hence, VA may be used as a stand-alone imaging device or as a tool to provide supplementary information to other existing imaging modalities. Materials & Methods: Matching vibro-acoustography and mammography images of human breast are acquired using a combined mammography/vibro-acoustography machine. Images of various types of breast masses are investigated and their characteristics features are compared. Image features, such as lesion borders, interior texture, and speculation pattern, are studied. Results: Resulting images show soft tissue structures, breast lesions and microcalcifications within breast with high contrast, high resolution, and no speckles. The results have been verified using x-ray mammograms of the breast. Vibro-acoustography images demonstrate that calcified masses normally show sharp borders, benign non-calcified masses have smooth texture, and, in some cases, malignant masses are characterized with spiculation. Conclusion: The encouraging results which are based on the data collected from an ongoing study indicate that the above mentioned image features may be used for differentiation of breast masses and suggest that further development of vibro-acoustography technology may lead to a new clinical tool for various imaging applications. Work supported in part by Grant CA121579-01A1from NIH and Grant BCTR#0504550 from Susan G. Komen Breast Cancer Foundation.

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 4047. doi:1538-7445.AM2012-4047
Abstract 4057: Quantitative assessment of 3D nuclear architecture in colon epithelial cells by micro-optical computed tomography
Authors: Nandakumar Vivek , Hernandez Kathryn F. , Rodolpho Beatriz L. , Senechal Patti , Bussey Kimberly J. , Johnson Roger H. , Meldrum Deirdre R.
Published: August 2012

Colorectal cancer (CRC) is a commonly diagnosed cancer and the third leading cause of cancer-related deaths in the United States. About 50,000 people will die from this disease in 2011. CRCs develop through an accumulation of genetic and epigenetic aberrations and have been broadly classified into three molecular subtypes - chromosomal instability (CIN), microsatellite instability (MSI), and CpG Island Methylator Phenotype (CIMP). Research studies on CRC have predominantly focused on its molecular biological and genetic basis, but very little is known about the three-dimensional cell morphological variations between these subclasses. We investigated alterations in the three-dimensional (3D) nuclear architecture of the three molecular subtypes of CRC, comparing them to a benign adenoma, using representative epithelial cell line models: AAC1 (benign), AAC1-SB10 (CIN), HCT-116 (MSI) and RKO (CIMP). We applied micro-optical computed tomography (Cell-CT) to image one hundred individual hematoxylin-stained cells of each of the four types with submicron, isotropic spatial resolution. We performed automated 3D image analysis on the volumetric cell images, quantifying fifty descriptors (features) to characterize the nuclear morphology and coarse chromatin organization. We evaluated the statistical efficacy of our 3D features in distinguishing among the cell types. Our results demonstrated previously unreported morphological variations among the controls and three types of CRC. Relative to the benign adenoma cells, in contrast to CIN and MSI, the CIMP subtype exhibited markedly smaller cell and nuclear sizes, smaller nucleus-to-cytoplasm (NC) ratio and a predominantly smooth nuclear interior, lacking in clumpy chromatin. However, the CIMP cell nuclei exhibited the highest optical density among the four cell classes. MSI cells showed the clumpiest nuclear texture, followed by the benign adenoma cell nuclei. These results offer new perspectives on nuclear structure variations in benign colon adenoma and colorectal cancer cells, warranting further investigation. They suggest that the hypermethylation seen in the CIMP phenotype may result in demonstrable chromatin compaction with a concomitant reduction in nuclear size. The chromatin clumpiness observed in the MSI cells may reflect increased mutations at nuclear scaffold binding sites, which tend to be characterized by single and di-nucleotide repeats. We intend to pursue studies on human biopsy cells with ultimate goal of testing the efficacy of Cell-CT for CRC diagnosis, subtyping and staging.

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 4057. doi:1538-7445.AM2012-4057
Abstract 406: Monitoring inflammation and therapeutic response with clinically translational 19F MRI
Authors: Helfer Brooke M. , Balducci Anthony , Wesa Amy
Published: August 2012

Non- invasive monitoring of inflammation to both diagnose disease and evaluate treatment is critically needed. V-Sense, a perfluorocarbon tracer agent that labels circulating inflammatory cells, which are then recruited out of circulation to sites of inflammation, enables detection by 19F MRI. Due to negligible 19F background in the host, detection of 19F and thus inflammation is highly-specific. Inflammation is a noted predecessor to several types of cancer such as colon, ovarian, pancreatic and prostate cancer and also contributes to the progression of metastatic disease. Using a collagen-induced arthritis model, where the inflammatory process is well understood, we demonstrate the ability to monitor development, progression, and treatment of inflammation. Disease progression in the rat hind limbs was monitored by caliper measurements and 19F MRI on days 14, 21 and 28, including the height of clinically symptomatic disease. Naïve rats served as controls. The capacity of V-Sense to assess the effectiveness of therapy was studied in a cohort of rats administered oral prednisolone on days 14 to 28. Terminal histopathological analysis was also performed. Comparison of caliper measurements to the 19F accumulation, from 19F MRI, validated the quanitative nature of this approach. This study supports the use of V-Sense to monitor and quantify the inflammatory process in disease, including the development of and treatment 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 406. doi:1538-7445.AM2012-406
Abstract 4062: A kinetic live-cell imaging approach to measuring cytotoxicity in vitro
Authors: OClair Belinda A. , Endsley Eric W. , Groppi Vince E. , Appledorn Daniel M.
Published: August 2012

Cytotoxicity assays play a critical role in the identification of potential anti-cancer chemotherapeutic agents. Commonly used in vitro cytotoxicity assays evaluate a range of end point parameters such as the release of lactate dehydrogenase (LDH) and glutathione (GSH) following membrane rupture, generation of reactive oxygen species (ROS), cell proliferation, and disruption of mitochondrial trans-membrane potential. Critical factors contributing to the predictive nature of these assays include compound concentration, and more importantly, the time allowed for the compound to elicit an effect. Although these multiplexed assays are able to simultaneously measure multiple indicators of in vitro cytotoxicity, they typically assess a single time point and are unable to assess the biological activity over time. We present data showing that we are able to kinetically measure cell wall integrity, while simultaneously monitoring cell death associated morphological changes using a commercially available cell impermeable DNA dye, YOYO-1, coupled with the IncuCyte™ FLR live-cell imaging system. This no-wash, mix-and-read assay provides a statistically robust method to study the kinetic induction of cytotoxicity in vitro. Specifically we use HT 1080 fibrosarcoma cells, MDA-MB-231 breast adenocarcinoma cells, and HeLa cervical adenocarcinoma cells in conjunction with the cytotoxic agents staurosporine and camptothecin, and the cytostatic agent, cycloheximide. These data illustrate the ability to kinetically detect, quantify, and analyze cytotoxicity data in a 96-well plate format using images collected in real time. We show that the quantitative data obtained in this study is amenable to moderate throughput screening protocols with Z’ values >0.6 achieved in both intra- and inter-assay reproducibility studies. We also provide evidence that that the high definition phase contrast images provide qualitative verification of quantitative results allowing for the discrimination between cytotoxic and cytostatic compounds. This strategy combines automated data acquisition and analysis to provide an informative and efficient approach to detecting pharmacological or genetic manipulations that alter cell viability.

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 4062. doi:1538-7445.AM2012-4062
Abstract 4071: PAXgene Tissue: A new tissue fixation technology for simultaneous preservation of morphology and nucleic acids
Authors: Grölz Daniel , Dettmann Nadine , Blassnig Isabell , Wyrich Ralf , Rainen Lynne
Published: August 2012

Introduction: PAXgene Tissue (PAXgene) is a new, formalin-free fixation technology developed for simultaneous preservation of morphology and biomolecules in tissue samples. PAXgene fixed, paraffin embedded (PFPE) tissue is suitable for conventional histochemical and immunohistochemical staining as well as for extraction of high quality nucleic acids. Material and Methods: Cases of human lung, breast and colorectal cancer were divided and fixed in formalin, PAXgene, or snap frozen in liquid nitrogen (LN2). PAXgene fixed, stabilized specimens were stored for up to four days at room temperature prior to processing. Formalin fixed, paraffin embedded (FFPE) and PFPE tissue morphologies were compared using H&E stain. RNA, miRNA and DNA were isolated from FFPE, PFPE and LN2 tissue. RNA expression was analyzed in 96-well RT-qPCR arrays with pre-designed assays, and miRNA expression profiling was performed with single Sybr-green or primer/probe based assays. DNA was analyzed by agarose gel electrophoresis, long-range, and multiplex PCR. Results: Morphology of PFPE samples was similar to or indistinguishable from FFPE tissue. A high correlation of both RNA and miRNA gene expression results was observed between PFPE and LN2 samples. For all PCR assays, Ct values were within +/− 2 Ct. In contrast, poor correlation was observed between RNA from FFPE compared to RNA from LN2 with differences of up to 10 Cts. Correlation of FFPE to LN2 was higher in the case of miRNA but was still not as high as that of miRNA from PFPE. High molecular weight DNA which performed well in PCR could be isolated from LN2 and PFPE tissue, but DNA from FFPE failed in long-range PCR and showed biased or no amplification in multiplexed PCR. Conclusion: The PAXgene Tissue fixation technology preserves morphology and nucleic acids in tissue samples. It enables morphological and multimodal research biomarker testing with one sample. PAXgene Tissue is for research use only. It is not intended for diagnostic use.

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 4071. doi:1538-7445.AM2012-4071
Abstract 4074: Prostaglandin E2 promotes intestinal tumor growth via DNA methylation
Authors: Xia Dianren , Wang Dingzhi , Kim Sun-Hee , Katoh Hiroshi , DuBois Raymond N.
Published: August 2012

Both chronic inflammation and DNA methylation have been implicated as early events during colorectal cancer (CRC) carcinogenesis. The use of nonsteroidal anti-inflammatory drugs (NSAIDs) reduces the risk for colorectal cancer (CRC) by 40-50%. NSAIDs exert some of their anti-inflammatory or anti-tumor effects by inhibiting prostaglandin-endoperoxide synthase (PTGS) 1 and 2. PTGS2-derived prostaglandin E2 (PGE2) is a pro-inflammatory mediator and promotes tumor progression. Promoter DNA methylation may promote tumor growth through silencing tumor suppressor or DNA repair genes. Whether chronic inflammation, specifically in this study the pro-inflammatory factor PGE2, plays a role in DNA methylation and contributes to CRC carcinogenesis is unknown. Here we demonstrated that PGE2 upregulated DNA methyltransferase (DNMT) 1 and 3B, which resulted to increased DNA methylation in the promoters of target genes O6-methylguanine-DNA methyltransferase (MGMT), cannabinoid receptor 1 (CNR1), CDKN2B, and MutL homolog 1 (MLH1) in CRC cell lines. Meanwhile, the expression of MGMT, CNR1, CDKN2B, and MLH1 were decreased. EP4 antagonist blocked the upregulation of DNMT1 and DNMT3B while knockdown of DNMT1 or DNMT3B attenuated the dwonregulation of MGMT, CNR1, CDKN2B, and MLH1 by PGE2 in LS-174T cells. In ApcMin/+ mice, PGE2 treatment also increased the expression of DNMT1 and DNMT3B and the DNA methylation of MGMT, CNR1, CDKN2B, and MLH1 promoters, and reduced their expression. All these alterations were accompanied by increases in both polyp number and size in the intestines of ApcMin/+ mice. More intriguingly, a demethylating agent 5-Aza-dC blocked the PGE2-induced DNA methylation and tumor growth in ApcMin/+ mice. And a combination treatment with a selective PTGS2 inhibitor celecoxib and 5-Aza-dC more effectively reduced the polyp number and size in ApcMin/+ mice in comparison with either single agent. Furthermore, the levels of PTGS2 and PGE2 correlated with the expression of DNMT1 and DNMT3B; The DNA methylation of MGMT, CNR1, and MLH1 correlated with the levels of PTGS2, PGE2, DNMT1, and DNMT3B in human colorectal carcinomas. These results suggest that PGE2 can silence tumor suppressor or DNA repair genes via DNA methylation to promote CRC carcinogenesis.

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 4074. doi:1538-7445.AM2012-4074
Abstract 4078: Antitumor effect of the histone deacetylase inhibitor MS-275 in combination with retinoic acid Am80 in human cutaneous T-cell lymphoma
Authors: Kato Yukihiko , Egusa Chizu , Maeda Tatsuo , Tsuboi Ryoji
Published: August 2012

Resistance to chemotherapy is a major hurdle in the treatment of cutaneous T cell lymphoma. Histone deacetylase (HDAC) inhibitors are known to display antitumor activity in different tumor types, including melanoma, and to reverse epigenetic repression of tumor suppressor genes, such as retinoic acid receptor ≤ (RARα). Some HDAC inhibitors have begun to be used for cutaneous T cell lymphoma patients in Japan. In this study, we tested the antitumor effect of the HDAC inhibitor MS-275 in combination with retinoic acid Am80 on two human cutaneous T cell lymphoma cell lines in vitro. Treatment of MS-275 showed a dose-dependent inhibitory effect on SeAx and MJ cell lines in a MTT assay. These cell lines were relatively resistant to Am80. A greater inhibitory effect (up to 80%) was achieved with a combination of MS-275 and Am80 compared with each agent alone. The lack of RARα2 gene expression was associated with histone deacetylation and gene methylation at the promoter level. Treatment with the combination of MS-275 and Am80 restored retinoid sensitivity by reversing RARα2 epigenetic silencing, which was verified by ChIP assay using histone H4. P21 was induced in both cell lines but the biological effect of MS-275 was G1cell cycle arrest in MJ and apoptosis in the SeAx cell line. These results suggest that the HDAC inhibitor MS-275 has a greater antitumor activity in combination with Am80 in cutaneous T cell lymphomas but that the degree of induced cell death may vary. The combination of HDAC inhibitors and retinoids represents a novel therapeutic approach for cutaneous T cell lymphoma that warrants clinical testing.

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 4078. doi:1538-7445.AM2012-4078
Abstract 4092: Cancer-type regulation of MIG-6 expression by 5-Aza-2′-deoxycytidine and Trichostatin A
Authors: Zhang Yu-Wen , Staal Ben , Vande Woude George F.
Published: August 2012

Mitogen inducible gene 6 (MIG-6) is a tumor suppressor gene located in chromosome 1p36 which is frequently lost in many human cancers. It is mainly viewed as a negative feedback regulator of epidermal growth factor receptor (EGFR) family members by directly binding to the receptors and thereby negatively regulating the signaling. Mice with Mig-6 deficiency are prone to develop epithelial hyperplasia or tumors in various tissues including lung, gallbladder, bile duct, uterus and skin. Previously, we reported a few mutations in MIG-6 gene in human lung cancer, but such event is rare. To determine if MIG-6 is regulated by epigenetic silencing, we identified a group of cancer cell lines with low or no MIG-6 expression and investigated the status of DNA methylation and histone deacetylation in MIG-6 promoter. We found that DNA methylation inhibitor 5-Aza-2′-deoxycytidine (5-Aza-dC) induced MIG-6 expression in the melanoma but not in the lung cancer cell lines, while histone deacetylase inhibitor Trichostatin A (TSA) induced MIG-6 expression in lung cancer lines but not in melanoma cell lines. However, the MIG-6 promoter itself does not seem to be directly affected by methylation or histone deacetylation, indicating they are indirectly regulated and most likely mediated by transcription factor(s). Luciferase reporter assays revealed that a short segment of exon-1 in MIG-6 gene is responsible for the induction of its expression by TSA in the lung cancer cells. Our data suggest that MIG-6 gene expression is differentially regulated by the inhibitors of methylation and histone deacetylation in lung cancer and melanoma cells respectively, without directly modifying the promoter.

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 4092. doi:1538-7445.AM2012-4092
Abstract 41: Identification of CXCL5/ENA-78 regulating the interaction of cholangiocarcinoma and cancer-associated fibroblasts
Authors: Okabe Hirohisa , Beppu Toru , Ueda Mitsuharu , Hayashi Hiromitsu , Masuda Toshiro , Mima Kosuke , Nakagawa Shigeki , Miyake Keisuke , Yokoyama Naomi , Baba Yoshifumi , Takamori Hiroshi , Ishiko Takatoshi , Jono Hirofumi , Shinriki Satoru , Ando Yukio , Baba Hideo
Published: August 2012

Background and Aims: Tumor-stromal interaction has been recognized to be essential for understanding tumor development. Cholangiocarcinoma shows very poor prognosis and have fibrous tumor stroma. Hepatic stellate cells are well-known to have a major role in hepatic fibrogenesis. Therefore, we have focused on the interaction of cholangiocarcinoma and hepatic stellate cells and reported their positive interaction (Ann Surg Oncol 2009, 2011). The aim of this study is to identify the key protein which is important for the interaction of cholangiocarcinoma cells and hepatic stellate cells and examine its functional role in tumor progression. Methods: Using co-culture model and proteomic analysis, we analyzed the secreted factors which were considered to be important for their interaction. The functional role of CXCL5 was examined in vitro and clinical significance was validated using human intrahepatic carcinoma samples by immunohistochemistry. Results: Using SELDI-TOF-MS and qTOF-MS/MS analysis, we identified CXCL5 which was remarkably increased when they were co-cultured. CXCL5 which was mainly produced by cholangiocarcinoma cell lines (HuCCT1 and RBE) promoted the invasion, and migration of them in the autocrine fashion. These effects were significantly decreased by inhibition of CXCR2 which was a receptor of CXCL5. IL1β was found to up-regulate in hepatic stellate cells when they were co-cultured by cytokine array analysis, we also found that IL1β accelerated the production of CXCL5 in cholangiocarcinoma cells. In human intrahepatic cholangiocarcinoma, immunohistochemical study showed that there was a significant correlation between stromal myofibroblasts and CXCL5 produced by cholangiocarcinoma cells. Since CXCL5 is identified as epithelial neutrophil activating factor-78 (ENA-78), we researched the presence of CD66b positive immune cells which have been recently reported to be tumor-associated neutrophils. CD66b positive immune cells are more abundant in CXCL5 high expression group. In addition, high CXCL5 expression group showed worse survival than low expression group. Conclusion: We conclude that CXCL5 is important for the interaction of cholangiocarcinoma and stromal myofibroblasts and that the strategies to inhibit tumor-stromal cell interactions may be beneficial in cholangiocarcinoma.

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 41. doi:1538-7445.AM2012-41
Abstract 4101: Prognostic significance of LINE-1 mathylation level in gastric cancer
Authors: Iwagami Shiro , Baba Yoshifumi , Watanabe Masayuki , Shigaki Hironobu , Miyake Keisuke , Kurashige Jyunji , Ishimoto Takatsugu , Iwatsuki Masayuki , Baba Hideo
Published: August 2012

Background: Genome-wide DNA hypomethylation plays a role in genomic instability and carcinogenesis. DNA methylation in the long interspersed nucleotide element-1, L1 (LINE-1) repetitive element is a good indicator of the global DNA methylation level. In some types of human neoplasms, the LINE-1 methylation level is attracting interest as a useful marker for predicting cancer prognosis. However, the prognostic significance of LINE-1 hypomethylaiton in gastric cancer remains unclear. Methods: Using 96 resected gastric cancer specimens, we quantified the LINE-1 methylation using bisulfite-pyrosequencing technology. A Cox proportional hazards model was used to calculate the hazard ratio (HR), adjusted for tumor stage. Results: Gastric cancers showed significantly lower LINE-1 methylation levels compared to matched normal gastric mucosa (p<0.0001; N=50). The distribution of the LINE-1 methylation level in the 96 cancers was as follows: mean, 71.0; median, 72.9; SD, 12.2; range, 28.0-97.5; interquartile range, 66.7-77.7 (all in 0-100 scale). LINE-1 methylation level was not associated with tumor stage, T status, or N status (all P>0.05). In univariate analysis, LINE-1 hypermethylation tumor (more than 77.7) tended to have longer overall survival (log-rank p=0.10; univariate HR=0.50, 95% CI 0.19-1.12, p=0.097), although that was not statistically significant. However, in stage-adjusted analysis, LINE-1 hypermethylation was significantly associated with overall survival (HR=0.38, 95% CI 0.14-0.87, p=0.021). Conclusions: LINE-1 hypermethylation in gastric cancer is associated with longer survival, suggesting that it has potential for use as a prognostic biomarker.

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 4101. doi:1538-7445.AM2012-4101
Abstract 4109: The unfolded protein response promotes tolerance to extreme hypoxia through autophagy dependent maintenance of cellular metabolism
Authors: Cojocari Dan , Weljie Aalim , Wen Jing , Rouschop Kasper M.A , Vellanki Ravi , Koritzinsky Marianne , Wouters Bradly G.
Published: August 2012

Oxygenation patterns in solid human tumors are highly heterogeneous both amongst and within tumors, and individual cells can be exposed to mild or extreme hypoxia. Hypoxia is known to influence the behavior of tumor cells in an adverse manner, resulting in poor response to radiotherapy, chemotherapy, and an increased metastatic capacity. The ability of cells to tolerate extreme hypoxia and the unusual metabolic environments found within tumors are not fully understood, but cell phenotype is influenced through at least three important oxygen-sensing pathways: HIF, mTOR and the unfolded protein response (UPR). Previously we have shown that the PERK-eIF2α arm of UPR, which is activated rapidly in response to extreme hypoxia, contributes to hypoxia tolerance in both cell lines and in human tumor xenografts. Furthermore, we found that signaling through PERK results in increased capacity for autophagy through direct transcriptional regulation of the autophagy genes ATG5 and LC3b during hypoxic exposure. We hypothesize that autophagy promotes survival during hypoxia through regulation of metabolic homeostasis and control of proteotoxicity. To elucidate the contribution of PERK signaling and autophagy to cell survival and metabolic homeostasis we created a series of isogenic cell lines with inducible expression of transgenes that cause PERK or autophagy signaling deficiency. We analyzed the changes in metabolism and in global metabolite levels during exposure to extreme hypoxia in wild type, PERK deficient, and autophagy deficient variants. Using liquid chromatography mass spectrometry, we identified a series of metabolites whose concentration varied in a time-dependent manner during hypoxia. Importantly, we found that cells defective in PERK signaling decreased levels of amino acids, increased fatty acids and evidence of oxidative stress products. Both, PERK signaling and autophagy deficiency resulted in accelerated glucose metabolism as evidenced by increased consumption and lactate production during hypoxia compared to controls. Together, these results suggest that PERK-dependent regulation of autophagy contributes to maintenance of bio-energetic status, preventing increased glucose flux and production of reactive oxygen species. Our data also demonstrate that aggressive, therapy resistant hypoxic cells may be selectively targeted by therapies that exploit the dependency of these cells on autophagy for 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 4109. doi:1538-7445.AM2012-4109
Abstract 4112: Prognostic impact of autophagic markers LC3, Beclin 1 and p62/sequestosome 1 in colon carcinomas from patients treated in 5-FU-based adjuvant therapy trials
Authors: Sinicrope Frank A. , Wu Tsung-Teh , Foster Nathan R. , Sargent Daniel J. , Huang Shengbing , Park Jae Myung
Published: August 2012

Background. Autophagy maintains energy metabolism and enables tumor cell survival during cellular stress. Beclin 1 is involved in autophagosome formation during which membrane-bound LC3 binds to p62/sequestosome 1. We reported that ectopic p62 can promote 5-fluorouracil-induced cell death (JBC 286:40002-12, 2011). It is unknown whether these autophagy markers can predict clinical outcome. Materials & Methods. Beclin 1, LC3B, and p62 proteins were analyzed by immunohistochemistry in tissue microarrays containing stage II and III colon carcinomas (N=151) from patients receiving 5-FU or surgery alone. The mean weighted score (intensity x extent) was dichotomized for survival analysis using Cox models. Results. Increased levels of cytoplasmic LC3, Beclin 1, and p62 expression were detected in 21%, 14%, and 68% of cancers, respectively, and were not associated with tumor site, stage or grade. By multivariable analysis, increased LC3 was significantly associated with worse disease-free survival (DFS) (p=0.022), but not overall survival (OS) [p=0.069](Table). Beclin 1 was not prognostic, but increased p62 was significantly associated with improved DFS (p=0.038), but not OS (p= 0.176)[Table]. When restricted to 5-FU-treated patients, increased LC3 was associated with worse DFS [HR= 2.38 (1.19, 4.75, p=0.020), elevated Beclin 1 predicted worse OS [HR=2.83 (1.27, 6.34), p = 0.019], and p62 was not prognostic. Conclusion. LC3 and Beclin 1 overexpression were independently associated with adverse outcome. Increased p62 was associated with better outcome consistent with its ability to promote cell death. These data support strategies to inhibit autophagy for therapeutic advantage.

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 4112. doi:1538-7445.AM2012-4112
Abstract 412: Expression of cancer stem cell markers in pancreatic ductal adenocarcinomas and pancreatic intraepithelial neoplasias
Authors: Kure Shoko , Matsuda Yoko , Hagio Masahito , Suzuki Taeko , Ueda Junji , Yamahatsu Kazuya , Yamamoto Tetsushi , Naito Zenya , Ishiwata Toshiyuki
Published: August 2012

Background: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy with a high incidence of distant metastasis. Recent studies have shown that cancer stem cells (CSCs) are important in cancer cell growth, invasion, metastasis, and recurrence. Several studies have revealed some CSC-specific markers for PDAC, such as CD133, CD24, CD44, CXCR4, ESA, and nestin. Some evidence also suggests that PDAC progresses through a multistep process comprised of noninvasive precursor lesions known as pancreatic intraepithelial neoplasias (PanINs). The reports of CSCs in PanINs are limited. We performed a comprehensive analysis of the expression of CSC markers in PDACs, PanINs, and normal pancreatic tissues. Materials & Methods: CD24, CD44, ESA, CD133, CXCR4, and nestin were used as CSC markers. Human PDACs (n=67), PanIN-1 (n=35), PanIN-2 (n=52), PanIN-3 (n=18), and normal pancreatic tissues (n=54), which were obtained from patients who underwent surgical operations in the surgical department of Nippon Medical School, were used for immunohistochemical analysis. Human PDAC cell lines, PANC-1, KLM-1, and MIA PaCa-2, were used for flow cytometric assays and quantitative RT-PCR analysis. Results: Regarding the clinicopathological features, the positivity of CD44 and CD133 showed significant correlations for UICC classifications and histological features In the survival analysis, CD133-positive PDACs led to a significantly poorer prognosis. In the immunohistochemical analysis, CD24, CD44, ESA, CXCR4, and nestin showed a gradual increase of positivity along with the grades of PanINs. Flow cytometric assays and RT-PCR analysis confirmed the expression profile in PDAC. Conclusion: CSC markers were expressed to various extents in PDAC, and the expression of CD133 and CD44 was correlated with the prognosis and stage. Furthermore, CSC markers showed a gradual increase along with the grades of PanINs; therefore, CSC may be involved in the carcinogenesis of PDAC.

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 412. doi:1538-7445.AM2012-412