bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4277: Development and validation of a novel IDH1-mutant astrocyte cell line as a model for high-grade gliomas
Authors: Robinson Nathaniel D. , Purshouse Karin R. , Fons Nathan R. , Breuer Gregory A. , Pusch Stefan , von Deimling Andreas , Sundaram Ranjini K. , Bindra Ranjit S.
Published: July 2016

High-grade gliomas (HGGs) are devastating malignancies of the central nervous system, and few treatment options are available for these tumors. In the most malignant form of the disease, glioblastoma multiforme (GBM), over 90% of patients will succumb to their tumor within 5 years after standard of care treatment, consisting of surgery, radiation therapy, and temozolomide chemotherapy. It is now clear that gliomas are molecularly heterogeneous entities, with mutations in tumor suppressors and oncogenes defining many distinct sub-types with important therapy implications. However, almost all HGGs are treated with a limited array of initial therapies, regardless of these molecular differences. Isocitrate dehydrogenase-1 (IDH1), a gene recently found to be mutated in many gliomas, is involved in the conversion of isocitrate to 2-oxoglutarate in cells. The IDH1 R132H mutant enzyme converts 2-oxoglutarate to the oncometabolite (R)-2-hydroxyglutarate (D2HG), which leads to profound metabolic alterations in tumor cells. In addition, recent studies indicate that mutations in IDH1 may also induce altered DSB repair, differential sensitivities to chemo-radiotherapy, and substantial changes in chromatin modifications. Here, we present the creation of a novel astrocyte cell line harboring an engineered heterozygous IDH1 R132H mutation at the endogenous gene locus using CRISPR/Cas9 gene editing. We confirmed expression of the engineered mutation at the protein level, and we have characterized this cell line in a comprehensive panel of functional assays. In particular, we demonstrated that our mutant cell clones secrete high levels of D2HG, and we confirmed that the levels of this oncometabolite can be suppressed with small molecule inhibitors of mutant IDH1. We also characterized the DNA damage response network in IDH1-mutant cells using high-content DNA damage foci assays recently developed by our group, and also in clonogenic survival assays. To our knowledge, this is the first report of an astrocyte cell line harboring an engineered, heterozygous R132H mutation at the endogenous locus. This novel cell line represents a new model system for studying gliomas and has tremendous applications for further cell characterization, mechanistic studies, and drug screening.

Citation Format: Nathaniel D. Robinson, Karin R. Purshouse, Nathan R. Fons, Gregory A. Breuer, Stefan Pusch, Andreas von Deimling, Ranjini K. Sundaram, Ranjit S. Bindra. Development and validation of a novel IDH1-mutant astrocyte cell line as a model for high-grade gliomas. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4277.
Abstract 4291: Vitamin B6 biomarkers and colorectal cancer: Modifications by time
Authors: Gylling Björn , Myte Robin , Schneede Jörn , Hallmans Göran , Häggström Jenny , Johansson Ingegerd , Ulvik Arve , Midttun Øivind , Ueland Per Magne , van Guelpen Bethany , Palmqvist Richard
Published: July 2016

Background: Pyridoxal 5’-phospate (PLP) is the most commonly used marker of circulating vitamin B6 status. In prospective case-control studies, individuals with higher plasma concentrations of PLP had a 30-50% decreased risk of colorectal cancer (CRC). This may partly be explained by decreased PLP due to inflammatory activity in persons at an increased risk of CRC. We aimed to investigate different markers of vitamin B6 in relation to CRC risk in a population with a very long follow-up time between screening and diagnosis.

Methods: This was a prospective case-control study of 613 incident CRC cases and 1190 matched controls nested within the population-based Northern Sweden Health and Disease Study. PLP, pyridoxal (PL), pyridoxic acid (PA), 3-hydoxykynurenine (HK) and xanthurenic acid (XA) were measured in plasma by LC-MS/MS. Besides PLP, we investigated the recently introduced metabolite ratios PAr (PA/(PLP+PL)) and HK:XA as markers of vitamin B6 status in relation to CRC risk.

Results: PAr, a marker of vitamin B6 catabolism during inflammation, was linearly associated with CRC risk, with a 50% higher risk in the highest vs. lowest quartile. In the group with a follow up time between screening and diagnosis over 10.5 years no association was observed, while in the groups with a follow up time between 5.8-10.5 years and below 5.8 years risk was approximately twofold in most quartiles vs the lowest quartile. A 50% risk decrease was observed in the in the third vs the lowest quartile of plasma PLP and a similar risk increase was observed in the highest vs the lowest quartile of the functional vitamin B6 marker HK:XA.

Conclusions: PAr may influence later stages of tumor development, but possibly not early tumorigenesis. Poor functional vitamin B6 status and inflammatory activity might influence both the risk of CRC and plasma concentrations of PLP. Our results underscore the need to use several biomarkers for vitamin B6 status together with the already well established plasma PLP. This may be generalizable not only to CRC but also other diseases with an inflammatory aspect.

Citation Format: Björn Gylling, Robin Myte, Jörn Schneede, Göran Hallmans, Jenny Häggström, Ingegerd Johansson, Arve Ulvik, Øivind Midttun, Per Magne Ueland, Bethany van Guelpen, Richard Palmqvist. Vitamin B6 biomarkers and colorectal cancer: Modifications by time. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4291.
Abstract 4314: Using antioxidant to enhance liver cancer preventive effect of n-3 PUFAs
Authors: Fu Ying , Bai Angela Y. , Dyba Marcin , Kang Jing X. , Chung Fung-Lung
Published: July 2016

In various animal models, n-3 polyunsaturated fatty acids (PUFAs) have demonstrated cancer preventive effects. This protective effect is associated with reduced levels of inflammatory cytokines, such as tumor necrosis factor alpha (TNF-α), cyclooxygenase 2 (COX-2) and prostaglandin E2 (PGE2). On the other hand, an endogenous DNA adduct formed from the peroxidation of ω-3 PUFAs, γ-hydroxy-1, N2-propanodeoxyguanosine (γ-OHPdG), is known to be mutagenic. In light of these observations, we hypothesized that the combination of n-3 PUFAs with an antioxidant to inhibit lipid peroxidation (e.g. α-lipoic acid) will result in an enhanced cancer preventive effect than n-3 PUFAs alone. The chemical carcinogen diethylnitrosamine (DEN) induced HCC model is employed in this study. The DEN-induced HCC model has a histology and genetic signature similar to that of human HCCs, such that it has poor prognosis and recapitulates a dependence on inflammatory signaling. It also reflects the same gender disparity seen in human HCCs. Fat-1 transgenic mice were used because they express a Caenorhabditis elegans desaturase converting n-6 to n-3 PUFAs endogenously without using dietary supplementation. This model underlies the importance of dietary control of PUFAs intake and ratios due to the inability of mammalian cells to generate anti-inflammatory n-3 PUFAs from pro-inflammatory n-6 PUFAs, which are a major component of the standard Western diets. The results showed that antioxidant can efficiently suppress the elevated level of γ-OHPdG in fat-1 mice. The tumor incidence, multiplicity, and size are monitored continuously. This work was supported by the NCI grant: RO1-CA-134892. Y.F. thanks the Prevent Cancer Foundation for his fellowship (Marcia and Frank Carlucci Charitable Foundation Award in Cancer Prevention and Early Detection)

Citation Format: Ying Fu, Angela Y. Bai, Marcin Dyba, Jing X. Kang, Fung-Lung Chung. Using antioxidant to enhance liver cancer preventive effect of n-3 PUFAs. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4314.
Abstract 4325: High-fat diet enhances and monocyte chemoattractant protein-1 deficiency reduces bone loss in mice with pulmonary metastases of Lewis lung carcinoma
Authors: Sundaram Sneha , Yan Lin
Published: July 2016

Bone is adversely affected by metastasis and metastasis-associated complications. Obesity is a risk factor for both bone and cancer. Adipose tissue is an endocrine organ that produces pro-inflammatory adipokines, such as monocyte chemotactic protein-1 (MCP-1), that contribute to obesity and obesity-related diseases. This study (2×2 factorial design) investigated the effects of a high-fat diet (45% of energy from corn oil) and MCP-1 deficiency on bone micro-structural changes, analyzed by micro-computed tomography, in male C57BL/6 mice bearing lung metastases of Lewis lung carcinoma (LLC). Subcutaneous injection of LLC cells resulted in lung metastasis which was significantly enhanced by the high-fat diet and attenuated by MCP-1 deficiency. Micro-computed tomographic measurement showed significant reductions in bone volume fraction (BV/TV), trabecular number (Tb.N) and bone mineral density (BMD) in right femurs and vertebrae of LLC-bearing mice compared to non-tumor-bearing controls. In LLC-bearing mice, compared to the low-fat diet (16% of energy from corn oil), the high-fat diet significantly reduced BV/TV, Tb.N and BMD in femurs but not in vertebrae. Furthermore, compared to wild-type mice, MCP-1 deficiency significantly increased BV/TV and Tb.N in both femurs and vertebrae and BMD in vertebrae in LLC-bearing mice. These results demonstrate that metastasis causes bone loss which is enhanced by the high-fat diet and attenuated by MCP-1 deficiency. Overall, it indicates that the high-fat diet and MCP-1 contribute positively to metastasis-associated bone deterioration.

Citation Format: Sneha Sundaram, Lin Yan. High-fat diet enhances and monocyte chemoattractant protein-1 deficiency reduces bone loss in mice with pulmonary metastases of Lewis lung carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4325.
Abstract 4334: A novel linker to enable alcohol-containing payloads for the preparation of antibody-drug conjugates
Authors: Kolakowski Robert V. , Haelsig Karl , Jeffrey Scott , Senter Peter
Published: July 2016

Antibody-drug conjugates (ADCs) have emerged as important therapeutics for treating cancer as evidenced by the recent FDA approval of brentuximab vedotin Adcetris™ and trastuzumab emtansineKadcyla™, and the large number of ADCs (>35) currently in clinical trials. ADCs are composed of two core units: the targeting antibody and the drug-linker. The drug-linker typically consists of a cytotoxic payload, a moiety for covalent attachment to the antibody, and a conditionally cleavable linker that is stable in circulation but releases the cytotoxic payload upon internalization into a target cell. The released payload will possess a residual chemical functional group that is used for attachment of the drug to the cleavable linker. Functional groups that have been exploited within the ADC field for this purpose include amines (primary and secondary), thiols, and carboxylic acids. Unfortunately, many potential ADC payloads do not possess any of these functional group ‘handles’ that allow for stable conjugation and release of the drug in an unmodified form. Use of these payloads often requires modification of the drug structure to introduce a handle for conjugation. To avoid this, we have developed new linker chemistry, the methylene-alkoxy-carbamate (MAC), which enables direct conjugation of drugs through alcohol functional groups. Such groups are present on a diverse array of synthetic drugs and cytotoxic natural products, thus, expanding the potential chemical space available for payload selection. As proof-of-concept, we have linked Auristatin E through the secondary alcohol of the norephedrine residue and characterized the stability and in vivo activity of the resulting ADCs. To further define the potential applicability of the MAC unit, we have examined its stability and release properties with a series of model systems including primary, secondary, and tertiary alcohols as well as a phenol. We have also explored the effect of substitution of the MAC nitrogen on these properties. This novel linker chemistry is currently being applied to new payloads that were inaccessible with previous technology.

Citation Format: Robert V. Kolakowski, Karl Haelsig, Scott Jeffrey, Peter Senter. A novel linker to enable alcohol-containing payloads for the preparation of antibody-drug conjugates. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4334.
Abstract 4338: Advanced neoplasia detection in colorectal cancer screening using multiple stool DNA markers and haemoglobin
Authors: Bosch Linda J.W. , Melotte Veerle , Mongera Sandra , Daenen Kathleen L.J. , Coupe Veerle H.M. , van Turenhout Sietze T. , Stoop Esther M. , de Wijkerslooth Thomas R. , Mulder Chris J.J. , Kuipers Ernst J. , Dekker Evelien , Domanico Michael , Lidgard Graham P. , Berger Barry M. , Carvalho Beatriz , van Engeland Manon , Meijer Gerrit A.
Published: July 2016

Purpose of the study: Molecular tests have the potential to improve current non-invasive faecal immunochemical test (FIT) screening for colorectal cancer (CRC) and advanced precancerous lesions. We examined the performance of a panel of faecal DNA (sDNA) markers and FIT in archival samples from an invitational CRC screening population.

Methods: Whole stool samples were prospectively collected from individuals participating an invitational primary colonoscopy-screening program (COCOS trial). Only participants that provided stool, performed FIT (OC-Sensor) and underwent colonoscopy were selected. The sDNA panel included quantitative molecular assays for KRAS mutations and for aberrant NDRG4 and BMP3 methylation. The performance of the sDNA plus FIT panel was compared to the FIT results alone, by Receiver Operator Characteristic (ROC) analyses.

Results: A total of 1047 individuals (51% male) with a median age of 60 years (range 50-75) were included, of which 7 (0.7%) had colorectal cancer and 104 (9.9%) had advanced precancerous lesions (advanced adenomas or sessile serrated polyps ≥ 1 cm).

The combination of sDNA and FIT was more sensitive than FIT alone for detecting advanced precancerous lesions (49% (50/102) and 25% (26/102), respectively). Specificities among individuals with non-advanced or negative findings (controls) were 89% and 96% for sDNA and FIT testing, respectively.

ROC analysis of CRC and advanced precancerous lesions compared to controls revealed an Area Under the Curve (AUC) of 0.75 for the sDNA plus FIT test, compared to 0.68 for FIT alone. At an equal specificity of 95%, advanced precancerous lesions were detected with a higher sensitivity by the sDNA plus FIT test compared to FIT alone (36% vs 28%, p = 0.08).

Conclusions: In an invitational colorectal cancer screening cohort, combining stool DNA markers with FIT detected more advanced neoplasia than FIT alone, primarily due to detecting more advanced adenomas.

Citation Format: Linda J.W. Bosch, Veerle Melotte, Sandra Mongera, Kathleen L.J. Daenen, Veerle H.M. Coupe, Sietze T. van Turenhout, Esther M. Stoop, Thomas R. de Wijkerslooth, Chris J.J. Mulder, Ernst J. Kuipers, Evelien Dekker, Michael Domanico, Graham P. Lidgard, Barry M. Berger, Beatriz Carvalho, Manon van Engeland, Gerrit A. Meijer. Advanced neoplasia detection in colorectal cancer screening using multiple stool DNA markers and haemoglobin. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4338.
Abstract 4348: A prospective study of smoking habit and risk of synchronous colorectal cancers
Authors: Drew David A. , Nishihara Reiko , Lochhead Paul , Kuchiba Aya , Rong Qian Zhi , Mima Kosuke , Nosho Katsuhiko , Wu Kana , Wang Molin , Spiegelman Donna , Giovannucci Edward L. , Fuchs Charles S. , Ogino Shuji , Chan Andrew T.
Published: July 2016

Background: Synchronous colorectal cancers (CRC) (2 or more distinct primary carcinomas simultaneously identified in one patient) may arise as a result of a “field effect” or shared etiologic factors. Synchronous CRC has been associated with somatic epigenetic changes; cigarette smoking has been associated with DNA methylation in CRC. Thus, we examined the association between cigarette smoking and risk of synchronous CRC.

Methods: Among men and women enrolled in the Health Professionals Follow-up Study and Nurses’ Health Study over 24 and 32 years, respectively, we examined the association of smoking with CRC. We examined the differential risk of developing synchronous CRC compared with solitary CRC, classified based on review of pathology reports, through duplication-method Cox proportional hazards regression.

Results: During 3,477,211 person-years of follow-up of 45,691 men and 88,614 women, we documented 1,960 individuals with solitary CRC and 45 individuals with synchronous CRC. The association of smoking with CRC differed significantly according to the presence of synchronous compared with solitary tumors (Pheterogeneity = 0.0008). Compared with never smokers, current smokers experienced a higher risk of synchronous CRC (multivariable HR = 5.26; 95% CI, 2.08-13.32). In contrast, current smokers did not experience an elevated risk of solitary cancer (multivariable HR = 0.97; 95% CI, 0.83-1.13). Similar differences in the association of cumulative pack-years smoked with CRC according to tumor synchronicity status were also observed (Pheterogeneity = 0.006). Smoking cessation for ≥10 years was significantly associated with reduced risk of synchronous tumors (multivariable HR = 0.44; 95% CI, 0.20-1.00), but not solitary tumors (multivariable HR = 1.07; 95% CI, 0.91-1.25) (Pheterogeneity = 0.003).

Conclusion: Current cigarette smoking is associated with an increased risk of synchronous but not solitary CRC. These data support a model in which smoking contributes to an etiologic field effect predisposing individuals to the development of synchronous CRC.

Citation Format: David A. Drew, Reiko Nishihara, Paul Lochhead, Aya Kuchiba, Zhi Rong Qian, Kosuke Mima, Katsuhiko Nosho, Kana Wu, Molin Wang, Donna Spiegelman, Edward L. Giovannucci, Charles S. Fuchs, Shuji Ogino, Andrew T. Chan. A prospective study of smoking habit and risk of synchronous colorectal cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4348.
Abstract 4354: CRISPR/Cas9 genome-wide gRNA library for target identification
Authors: Tedesco Donato , Diehl Paul , Makhanov Mikhail , Baron Sylvain , Suchkov Dmitry , Frangou Costa , Chenchik Alex
Published: July 2016

Genome-wide loss-of-function screening is a fundamental method to identify genes responsible for driving biological responses, and complex pooled lentiviral-based libraries expressing large numbers of genetic disruptors, such as shRNAs, make large-scale cell screening practical. While RNAi-based approaches have proven to be an effective strategy for these screens, recent work suggests CRISPR technology offers an effective alternative. Although shRNA and sgRNA pooled library screens are similar in concept, the gene interruption with the two techniques occurs by a very different mechanism so some divergence may be expected when comparing results obtained using one method versus the other.

To investigate the potential difference in the two methodologies, we performed parallel dropout viability screens to identify essential genes in a pair of primary isogenic CML cell lines using a CRISPR/Cas9 knockout library and an RNA interference (RNAi) library targeting the same set of 6,300 genes with the same number of targeted effectors (sgRNA or shRNA) for each gene. The results showed significant, but not complete, overlap in the essential genes identified by each assay in each cell line indicating that both approaches are effective to identify the majority of essential genes in a cell system. However, analysis did indicate that a small number of essential targets were only identified with CRISPR and certain unique targets seemed to show up only in the RNAi screen results. By combining data from the two screening methodologies, a consistent number of viability genes and pathways could be identified and subsequently validated by independent cell based assays at a very high confirmation rate.

Citation Format: Donato Tedesco, Paul Diehl, Mikhail Makhanov, Sylvain Baron, Dmitry Suchkov, Costa Frangou, Alex Chenchik. CRISPR/Cas9 genome-wide gRNA library for target identification. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4354.
Abstract 4378: Discovery and characterization of two novel drivers of hepatocellular carcinoma
Authors: Feddersen Charlotte R. , Dupuy Adam J.
Published: July 2016

Liver cancer is globally the sixth most commonly diagnosed and the third most deadly cancer. Hepatocellular carcinoma (HCC) is the most common form of the disease and a difficult cancer to treat due to inadequately understood pathways involved in tumorigenesis and progression. To obtain a better understanding of key HCC driver mutations, we performed a forward genetic screen using Sleeping Beauty transposon mutagenesis in a damaged liver microenvironment. Briefly, T2Onc3; Rosa26-SB11lsl/lsl;Alb-Cre mice were treated with carbon tetrachloride (CCl4) during ongoing transposition. We then sequenced the resulting liver tumors using linker mediated PCR (LM-PCR) and Illumina sequencing. We analyzed the sequenced data using gene-centric common insertion site (gCIS) analysis that allowed us to determine common insertion sites across tumors. These common insertion sites are referred to as candidate driver mutations.

This screen demonstrated the role of Glioma associated protein-2 (Gli2) and Fidgetin (Fign) as candidate driver mutations in a significant subset of HCC tumors (20% and 10% respectively). Gli2 is a key member of the Hedgehog signaling pathway, acting as a transcription factor for genes promoting cell proliferation. Fign is a relatively uncharacterized protein in the AAA-ATPase family with the ability to sever microtubules in vitro. We validated Gli2 as a driver of HCC by generating a hepatocyte specific Gli2-overexpression mouse model using hydrodynamic tail vein injection of Gli2 cDNA into FAH-/- mice. Mice underwent rapid tumorigenesis, regardless of CCl4 treatment, demonstrating that Gli2 is a strong oncogene. Additionally we have demonstrated that overexpression of Fign drives cell proliferation and promotes stabilization of the primary cilium in hepatocytes, a novel finding that provides insight into the mechanism of Fign-driven HCC.

The discovery of two novel HCC candidate driver mutations, validation of Gli2 as a bona fide HCC oncogene, and mechanistic insight into these commonly mutated genes in HCC will allow for future research into understanding HCC tumorigenesis and potential therapeutic options.

Citation Format: Charlotte R. Feddersen, Adam J. Dupuy. Discovery and characterization of two novel drivers of hepatocellular carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4378.
Abstract 4381: High-throughput in vivo systems biology using implantable microdevices
Authors: Jonas Oliver
Published: July 2016

We have developed an implantable device that can perform drug sensitivity testing of a large number of anti-cancer drugs inside the living tumor. This device contains reservoirs which release microdoses of single agents or drug combinations into distinct spatial regions of the tumor in local concentrations that are representative of concentrations achieved during systemic drug treatment. The efficacy of each of up to 30 individual drugs or combinations can be assessed from a single specimen without systemic drug exposure through minimally invasive biopsy. This assay takes into consideration physiologic effects that contribute to drug response by allowing drugs to interact with the living tumor in its native microenvironment.

Using this technology, we have been able to assess phenotypic drug response for up to 100 therapies in a single mouse, probing all clinically relevant single and combination treatments related to the estrogen receptor, CDK4/6 and PI3K pathways in ER+ PDX mouse models. We also examined a range of targeted and cytotoxic drug therapies in parallel before, during and after systemic drug treatments to investigate emerging drug resistance at the phenotypic level in BRAF-mutant melanoma models. The results demonstrate new patterns of escape and adaptive signaling mechanisms, and may offer clues for effective combination therapy.

Citation Format: Oliver Jonas. High-throughput in vivo systems biology using implantable microdevices. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4381.
Abstract 440: Quantification of somatic chromosomal rearrangements in circulating cell-free DNA from ovarian cancers
Authors: Harris Faye R. , Kovtun Irina , Smadbeck James , Multinu Francesco , Jatoi Aminah , Kalli Kimberly R. , Murphy Stephen J. , Halling Geoffrey C. , Kosari Farhad , Johnson Sarah H. , Mariani Andrea , Vasmatzis George
Published: July 2016

Circulating tumor DNA (ctDNA) provides great potential for the non-invasive clinical assessment of tumor burden. We developed a monitoring protocol using next generation mate-pair sequencing to identify chromosomal rearrangement signatures in primary tumors to follow ctDNA in blood. Primary tumors of 10 patients with ovarian cancer were sequenced, and individualized tumor-specific panels were designed to detect the junctions in ctDNA. A novel algorithm was developed and applied to data generated by quantitative PCR to calculate the percent of ctDNA. Selected junctions, some with potential as therapeutic targets, were detected in pre-surgically drawn blood in eight out of ten patients. Of these eight, three demonstrated the continued presence of circulating tumor DNA post-surgery, and five had undetectable levels, consistent with their documented presence or absence of disease. The detection of somatic junctions derived from ctDNA could be an effective way to monitor ovarian cancer patients for relapse and therapy response.

Citation Format: Faye R. Harris, Irina Kovtun, James Smadbeck, Francesco Multinu, Aminah Jatoi, Kimberly R. Kalli, Stephen J. Murphy, Geoffrey C. Halling, Farhad Kosari, Sarah H. Johnson, Andrea Mariani, George Vasmatzis. Quantification of somatic chromosomal rearrangements in circulating cell-free DNA from ovarian cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 440.
Abstract 4403: SMYD2-mediated lysine methylation regulates nuclear transport of beta-catenin in human cancer cells
Authors: Deng Xiaolan , Nakamura Yusuke , Hamamoto Ryuji
Published: July 2016

The canonical Wnt-signal pathway is a major signaling system in cancer cells as well as stem/progenitor cells. It is well-known that binding of Wnt molecules to cell surface receptors, including a Frizzled (Fz) protein and LDL receptor-related protein 6, initiates a transduction cascade leading to stabilization of the transcription coactivator β-catenin, which then enters the nucleus to form a transcriptional complex with T cell factor (TCF) or lymphoid enhancer factor (LEF) to activate the expression of TCF/LEF downstream genes, such as cyclin D1 (CCND1) and c-Myc. However, the molecular mechanisms that regulate nuclear localization of β-catenin and its transcriptional activation are not fully understood. Here we demonstrate that the protein lysine methyltransferase SMYD2 methylates β-catenin at lysine 133, which is located in an amino-terminal domain (NTD), and regulates nuclear transport of β-catenin. After treatment with SMYD2-specific siRNAs in hepatocellular carcinoma SNU475 and SNU449 cells, we fractionated cell components and conducted western blot analysis and found that nuclear localization of β-catenin was significantly decreased after knockdown of SMYD2; we also confirmed this result by immunocytochemistry. In addition, we prepared a lysine 133-substituted β-catenin mutant expression vector and transfected it into 293T cells with a SMYD2 expression vector. We observed that protein amount of lysine 133-substituted β-catenin in the nucleus is significantly lower than that of wild-type β-catenin by western-blot and immunocytochemical analyses. These results indicate that SMYD2-mediated methylation plays a critical role in the nuclear transport of β-catenin in cancer cells. This is a novel mechanism of the canonical Wnt-signal pathway regulated by lysine methylation.

Citation Format: Xiaolan Deng, Yusuke Nakamura, Ryuji Hamamoto. SMYD2-mediated lysine methylation regulates nuclear transport of beta-catenin in human cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4403.
Abstract 4407: Inhibition of STAT3 in primary trastuzumab-resistant HER2-positive cells leads to decrease cell viability
Authors: Helu Xousaen M. , Fernandez Alfida , De Leon Daisy D.
Published: July 2016

Breast Cancer is the second leading cause of cancer related death in women. Approximately, 20%-30% of breast cancer patients overexpress Human Epidermal Growth Factor Receptor 2 (HER2). HER2 Is a Tyrosine Kinase receptor involved in proliferation and survival and its overexpression makes it a good target for patients with HER2 positive breast cancer. Trastuzumab (TRA) is a recombinant monoclonal antibody that binds HER2 receptor and inhibits its signaling. Unfortunately, clinical studies have demonstrated that 30% of patients that overexpress HER2 do not respond to Trastuzumab treatment. Therefore, understanding the molecular mechanisms underlying Trastuzumab resistance is essential to develop new therapies to treat these breast cancer patients. Previous studies in our lab demonstrated that a decrease in cell growth of HER2 overexpressing cells occurred when IGF2 and HER2 were inhibited. Nevertheless, these cells overcame the growth inhibition and the resulting proliferative growth coincided with an increase in STAT3 activation. STAT3 is a transcription factor that promotes cell proliferation and can regulate apoptosis in cancer cells. These findings lead us to hypothesize that blocking STAT3 together with IGF2 and HER2 will inhibit proliferation and cell viability of Trastuzumab resistant breast cancer cells. We treated Trastuzumab-resistant HER2 positive JIMT1 cells with HER2, IGF2, and/or STAT3 inhibitors. After 24 hours of treatment, there was a marked difference in cell morphology observed between the treatment groups. Cell viability was measured using WST-1, a reagent that measures mitochondrial activity. A significant decrease in cell viability was observed when cells were treated for 24 hours with a commercially available STAT3 inhibitor (Sttatic). There was no difference between the treatment groups when STAT3 was inhibited by Stattic or when STAT3 was inhibited in combination with IGF2 and/or HER2 inhibitors. These findings suggest that in Trastuzumab-resistant HER2 positive JIMT1 cells, STAT3 activation overcomes the inhibition of the HER2 and IGF2 proliferation pathways resulting in cell death prevention. Our study demonstrate that Trastuzumab-resistant HER2 positive JIMT1 cells can activate STAT3 as an alternative pathway to evade apoptosis and promote chemoresistance. Our data also illustrate the challenge of treating cancer based on single therapies since tumors have many alternative pathways to promote growth.

Citation Format: Xousaen M. Helu, Alfida Fernandez, Daisy D. De Leon. Inhibition of STAT3 in primary trastuzumab-resistant HER2-positive cells leads to decrease cell viability. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4407.
Abstract 441: Association of interferon inducible genes with tumor immune microenvironment and chemotherapy resistance in high-grade serous epithelial ovarian cancer
Authors: Au Katrina K. , Meunier Liliane , Le Page Cécile , Graham Charles H. , Craig Andrew WB , Childs Timothy , Francis Julie-Ann , Squire Jeremy , Mes-Masson Anne-Marie , Koti Madhuri
Published: July 2016

Chemotherapy resistance is a major hurdle in high-grade serous epithelial ovarian cancer (HGSC) management. We previously reported differential expression of interferon inducible genes in pre-treatment tumours from chemoresistant and sensitive HGSC tumors. STAT1 expression was evaluated as a prognostic and predictive biomarker via immunohistochemistry in Phase I (n = 183) and Phase II (n = 550) biomarker validation studies conducted on HGSC tumours accrued from the Terry Fox Research Institute- Canadian Ovarian Experimental Unified Resource (TFRI-COEUR). Tumor STAT1 expression levels significantly correlated with the density of tumor infiltrating CD8+ T lymphocytes in both Phase I and Phase II cohorts. STAT1 expression significantly associated with progression free survival and response to chemotherapy in both Phase I and Phase II validation studies. Significant positive correlation between STAT1 expression levels and intratumoral CD8+ T cell density was observed. Intratumoral CD8+ T cell infiltration did not associate with progression free survival or response to chemotherapy in both cohorts. Interestingly, the prognostic relevance of CD8+ T cell was enhanced in combination with STAT1 in both cohorts. These findings provide evidence that STAT1 as an independent biomarker and a combination of CD8+ T cell infiltration with STAT1 could be novel immune-based prognostic and predictive biomarkers in HGSC. Findings from the current study will aid in patient stratification for novel immunomodulatory therapies for the management of chemotherapy resistance in HGSC.

Citation Format: Katrina K. Au, Liliane Meunier, Cécile Le Page, Charles H. Graham, Andrew WB Craig, Timothy Childs, Julie-Ann Francis, Jeremy Squire, Anne-Marie Mes-Masson, Madhuri Koti. Association of interferon inducible genes with tumor immune microenvironment and chemotherapy resistance in high-grade serous epithelial ovarian cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 441.
Abstract 4420: Heparan sulfate proteoglycans mediate SW13 oncogenic potential and response to granulin
Authors: Davis McKale , Hull Elizabeth
Published: July 2016

The SW13 adrenal adenocarcinoma cell line exists as two epigenetically and phenotypically distinct subtypes. One is a highly proliferative epithelial subtype lacking the expression of key tumor suppressor proteins, Brm and Brg1, the intermediate filament protein vimentin, and the cell cycle check point protein CD44 (SW13-), while the other is a slow growing, mesenchymal-like subtype which expresses all of these proteins (SW13+). Evidence strongly suggests that subtype determination is epigenetic in nature, as HDAC inhibitors can induce an SW13- to SW13+ subtype switch, but the cellular and environmental factors that regulate cellular phenotype have yet to be characterized. We performed a comprehensive microarray analysis and identified heparan sulfate proteoglycan (HSPG) binding as a primary pathway affected between these two subtypes. HSPGs are glycoproteins present in the extracellular matrix that can have tumor-promoting or tumor-suppressing activities depending on the number and sulfation status of attached sugar chains known as glycosaminoglycans (GAGs). Quantitative RT-PCR was then used to confirm the increased expression of the HSPGs, serglycin and glypican-3, as well as the HSPG-binding growth factors granulin and fibroblast growth factors-1 and -2 in SW13+ cells. Furthermore, HDAC inhibition was also shown to increase the expression of these genes in a time-dependent manner. As granulin over-production is associated with cancer progression in a number of human cancers, we were interested in examining the role of this HSPG binding growth factor further. A pro-granulin ELISA showed that SW13+ cells secrete significantly more granulin than SW13- cells. Intriguingly however, treatment with exogenous granulin increases growth rates in the already highly proliferative SW13- subtype, but had no effect on SW13+ growth. We hypothesized that the pleiotropic response of SW13 cells to growth factor treatment may be the result of HSPG post-translational modifications, so we assessed total GAG sulfation was by dimethylmethylene blue assay, and indeed the SW13+ cells were found to have significantly higher levels of total GAG sulfation than SW13- cells. Thus, transcriptional and post-translational modifications of HSPGs may contribute to SW13 phenotype control, and ultimately may affect tumorigenesis.

Citation Format: McKale Davis, Elizabeth Hull. Heparan sulfate proteoglycans mediate SW13 oncogenic potential and response to granulin. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4420.
Abstract 4421: Molecular features of hepatocellular carcinoma arising from non-alcoholic fatty liver
Authors: Takeda Makoto , Sakaguchi Takanori , Kiuchi Ryouta , Hiraide Takanori , Shibasaki Yasushi , Morita Yoshihumi , Kikuchi Hirotoshi , Setou Mitsutoshi , Konno Hiroyuki
Published: July 2016

(Background)

The incidence of hepatocellular carcinoma (HCC) arising from non-alcoholic fatty liver (NAFL) has been recently increasing according to increase of patients having metabolic syndrome. HCC, which shows steatosis, cellular ballooning, Mallory-Denk bodies, inflammation and fibrosis in cancer tissue, are recognized as steatohepatitic HCC (SH-HCC). This HCC phenotype is more frequently found in NAFL patients than in viral-hepatitic patients.

(Purpose)

The purpose of this study is to elucidate molecular features of SH-HCC with NAFL background.

(Methods)

To profile SH-HCC-related genes, mRNA expression patterns in SH-HCCs were compared with those in viral hepatitis HCCs by RNA array. RNA expression of the identified SH-HCC related gene was analyzed in three human liver carcinoma cell lines (HepG2, Huh7, and HLE) in normal conditioned medium. By mimicking the lipid-rich environment, these cell lines were also cultured in the presence of saturated (palmitic or stearic acid, recognized as endoplasmic reticulum stressors) or unsaturated fatty acids (oleic acid: OA). To address the role of SH-HCC-related gene, cell proliferation and apoptosis were assessed in the presence or absence of fatty acids, by knock-down of the objective gene using siRNA.

(Results)

(1) Caveolin (cav)-1 and -2 were identified as SH-HCC related genes (1.63 and 1.58 fold expression of cav-1 and -2, respectively) by RNA microarray analysis.

(2) HLE and Huh7 showed high and low expression of both cav-1 and -2 mRNA, whereas HepG2 did not express either on qRT-PCR.

(3) In the presence of OA, HepG2 and Huh7 did not well proliferate, whereas HLE cell proliferation increased with concentration of OA and culture duration.

(4) Knock-down of cav-1 and -2 canceled OA-induced increase of cell proliferation. In the same condition, expression of cleaved caspase 3 was increased, suggesting the promotion of apoptosis.

(Consideration)

Cav-1 and -2, the important protein for endocytosis and cellular signaling, are highly expressed in SH-HCC. These proteins would increase cell proliferation and inhibit apoptosis in the presence of unsaturated fatty acids, presumably by using these fatty acids as energy source or by caveolae-related anti-apoptotic process.

Citation Format: Makoto Takeda, Takanori Sakaguchi, Ryouta Kiuchi, Takanori Hiraide, Yasushi Shibasaki, Yoshihumi Morita, Hirotoshi Kikuchi, Mitsutoshi Setou, Hiroyuki Konno. Molecular features of hepatocellular carcinoma arising from non-alcoholic fatty liver. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4421.
Abstract 4437: DNA methylation analysis in cisplatin resistant ovarian cancer cells and recurrent ovarian cancer patient samples
Authors: Granados Michaela L. , Hudson Laurie G. , Luo Li , Samudio-Ruiz Sabrina L.
Published: July 2016

Acquisition of platinum resistance following first line platinum therapy is commonly observed in ovarian cancer patients and is a major obstacle to clinical effectiveness. Currently there are no options available to prevent platinum resistance; however, studies show that demethylating agents may resensitize patients to platinum therapy thereby demonstrating DNA methylation as a critical contributor to the development of platinum resistance. Using a cellular model of platinum resistance we have previously shown that resistant cells have increased DNA methyltransferase (DNMT) activity as well as increased global DNA methylation. These observations were dependent on cisplatin induced activation of the Epidermal Growth Factor Receptor (EGFR) which we previously identified as a novel a regulator of DNMT activity and DNA methylation. Inhibition of the EGFR conferred during a platinum resistance paradigm decreased DNMT activity, decreased global methylation and attenuated resistance in our platinum resistant model. We hypothesized that further genome wide analysis of DNA methylation in resistant, parental and EGFR inhibited cell lines at specific CG sites would reveal targets of methylation important for the development of platinum resistance. Using the Illumina 450K methylation array, we found few significant alterations in DNA methylation for given genes between control and cisplatin resistant (CPR) cells. However, additional DNA methylation analysis with the 450K methylation array of patient tumor samples at initial diagnosis as well as after relapse, while not significant, showed similar trends in the data for certain genes namely CSRNP3, LOC400904, LYPD6, MTMR9L, NIPAL4 and RBP7. We are still evaluating the effects of EGFR inhibition in vitro on DNA methylation patterns. We suggest that while the in vitro model of platinum resistance corresponds to patient samples in some aspects, ovarian cancer cells display a great deal of heterogeneity in DNA methylation as described by others and a much larger sample size is necessary to draw concrete conclusions. Current studies are evaluating gene expression in control and resistant cells to validate whether alterations in DNA methylation coincide with changes in expression.

Citation Format: Michaela L. Granados, Laurie G. Hudson, Li Luo, Sabrina L. Samudio-Ruiz. DNA methylation analysis in cisplatin resistant ovarian cancer cells and recurrent ovarian cancer patient samples. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4437.
Abstract 4449: TET1 inhibits gastric cancer metastasis by demethylation and re-expression of PTEN
Authors: Liu Bingya , Pei Yao-fei , Li Jian-fang , Su Liping , Tao Ran , Yan Min , Zhu Zhang-gang
Published: July 2016

TET1 is a member of ten eleven translocation enzymes, which can convert 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC), eventually to promote CpG islands demethylation of specific gene.. In this study, we found that TET1 expression and 5-hmC content were low in tumor tissues compared to its adjacent non-tumor tissues of gastric cancer. Cell proliferation, migration and invasion were enhanced when TET1 knocked down in gastric cancer cell in vitro and in vivo, while it was suppressed when TET1 over-expressed. We also found that PTENis one of the target genes of TET1. TET1 directly binds to the promoter region of PTEN, activates its transcription through demethylating its CpG islands. Knockdown of TET1 results in hypermethylation of PTEN promoter and low expression of PTEN. PTEN knockdown activates AKT and FAK pathway, which are suppressed by PTEN. The activation of PTEN downstream AKT and FAK facilitates tumor migration, invasion and accelerates cell growth. In conclution, we found a novel mechanism that TET1 suppresses tumor cell growth, migration and invasion through conversion PTEN promoter CpG island methylation to demehylation and then re-expression PTEN.

Citation Format: Bingya Liu, Yao-fei Pei, Jian-fang Li, Liping Su, Ran Tao, Min Yan, Zhang-gang Zhu. TET1 inhibits gastric cancer metastasis by demethylation and re-expression of PTEN. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4449.