bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2412: The modulatory effects of H. pylori infection in the DNA mismatch repair
Authors: Santos Juliana C. , Almeida Victor R. de , Fischer Wolfgang , Haas Rainer , Ribeiro Marcelo L.
Published: May 2015

The bacterial pathogen H. pylori chronically infects the human gastric mucosa and is the leading risk factor for the development of gastric cancer. It is believed that during the infection the organism causes an impairment of DNA repair in the gastric epithelium. Thus, we evaluated the in vitro modulatory effects of H. pylori infection in the DNA mismatch repair (MMR).

To evaluate the effects of H. pylori on MMR, AGS gastric carcinoma cell line and the H. pylori strain P12 were used in co-culture experiments for 4, 8 and 12 h. In addition, to analyze the potential effects of virulence factors on MMR, the ΔcagPAI, ΔvacA, ΔbabA, ΔhopQ, and Δtfs4 mutants were also subjected to co-culture experiments. The expression levels of EXO1, MLH1, MSH2, MSH3, MSH6, POLD3, PMS1, and PMS2 were assessed by real-time PCR. Moreover, expression levels of five previously screened miRNAs (miR-142-5p, miR-150-5p, miR-155-5p, miR-3163, and miR-4775), which are predicted to target MMR genes, were also evaluated by real-time PCR.

Our data show that H. pylori infection significantly down-regulates the expression of all selected genes. Regarding the virulence factors, the results suggest that the modulation on gene expression might be dependent on cagPAI, vacA, and the type IV secretion (TFS4) system. Although the infection up-regulates miR-150-5p, miR-155-5p, miR-3163, and miR-4775, only the expression of miR-155-5p is related with virulence factors.

Briefly, this study shows that H. pylori regulates the expression of several MMR genes, and it may be related with cagPAI, vacA, and the TFS4 system. Taking into account that MLH1, MSH2, and MSH6 are the miR-155-5p predicted targets, these preliminary data suggest that the observed virulence factor-dependent repression of miR expression could be responsible for the down-regulation of these MMR genes.

Citation Format: Juliana C. Santos, Victor R. de Almeida, Wolfgang Fischer, Rainer Haas, Marcelo L. Ribeiro. The modulatory effects of H. pylori infection in the DNA mismatch repair. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2412. doi:10.1158/1538-7445.AM2014-2412
Abstract 2416: Protein expression analysis of premature termination codon containing mutant proteins in colon cancers with high microsatellite instability
Authors: Yoon SeongJu , Kim Won Kyu , Park Jeon Han , Kim Hoguen
Published: May 2015

Frameshift mutations at coding mononucleotide repeats (cMNR) are common in the tumors with high microsatellite instability (MSI-H). These mutations invariably generate mRNAs containing abnormal coding sequences and premature termination codons (PTC). Normally, these mRNAs containing PTC are degraded by nonsense mediated mRNA decay (NMD) system. However, mRNAs containing PTC located in the last exon are not subject to be degraded by NMD system, and thus expected to generate truncated mutant proteins. We identified 29 novel mutations at coding region mononucleotide repeats (cMNR) genes by a PCR based method and sequencing. The frequency was varying (10% to 90%) in the 10 MSI-H colon cancer cell lines. To confirm the mRNA level, we analyzed the mutant mRNAs from identified genes in colon cancer cell lines and found that the mutant mRNAs were not degraded in cells. For mutant protein detection, we adopted both immunobloting and a target mass method to detect endogenous mutant proteins in cells. By immunobloting analysis, we found rare level of endogenous mutant proteins in cells. Using a target mass method, however, we could detect potential mutant protein candidates and confirmed one of the mutant candidates (VPS37B mutant proteins) is stably expressed in cells by in vitro overexpression experiments. When mutated, VPS37B mutant proteins came to acquire very short neopeptides (only two novel amino acids) which mainly disturb the stable expression of mutant proteins and this might explain the intact expression of VPS37B mutant proteins. Taken together, our findings suggest that frameshift mutations at cMNR of genes usually lead to the rare expression of the mutant proteins in tumor cells but, some mutant proteins with short neopeptides could be stably detected by a highly sensitive target mass method.

Citation Format: SeongJu Yoon, Won Kyu Kim, Jeon Han Park, Hoguen Kim. Protein expression analysis of premature termination codon containing mutant proteins in colon cancers with high microsatellite instability. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2416. doi:10.1158/1538-7445.AM2014-2416
Abstract 2419: Protective role of thioredoxin-1 in base excision repair under redox modulation
Authors: Kim Hye Lim , Koedrith Preeyaporn , Lee Sang Min , Kim Yeo Jin , Seo Young Rok
Published: May 2015

Thioredoxin-1 (Trx1) is an antioxidant enzyme with a protective role in the removal of reactive oxygen species (ROS). We investigated the mechanism by which the redox modulator Trx1 affects base excision repair (BER) activity to understand the protective role of Trx1. We constructed a Trx1 knockdown system to demonstrate the specific mechanism of Trx1. DNA damage in terms of relative intensity of the DNA tail and γ-H2AX foci was markedly higher in the Trx1 shRNA cells compared with that in the wild type cells, leading to increased cellular susceptibility to a sublethal dose of BER-inducible toxicant, nitrosomethylurea (NMU). In addition, we observed a modulatory role of Trx1 in the BER pathway via the p53 downstream gene, growth arrest, and DNA-damage-inducible protein 45 α (Gadd45a). The protein level and function of p53, a Trx1 downstream gene, coincidently decreased in the Trx1 shRNA cells. Furthermore, Trx1 shRNA cells showed decreased Gadd45a expression and interaction of Gadd45a with apurynic/apyrimidinic endonuclease 1 (APE1) as well as APE1 activity. In conclusion, Trx1 might cooperate in the control of APE1 function by modulating the p53-mediated BER via the protein-protein interaction between Gadd45a and APE1, providing insight into the novel role of redox factor Trx1 in modulation of BER.

Note: This abstract was not presented at the meeting.

Citation Format: Hye Lim Kim, Preeyaporn Koedrith, Sang Min Lee, Yeo Jin Kim, Young Rok Seo. Protective role of thioredoxin-1 in base excision repair under redox modulation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2419. doi:10.1158/1538-7445.AM2014-2419
Abstract 242: Dietary PUFA's alter the visceral adipose secretome, which modulates mammary stem cell self-renewal
Authors: Esper Ray , Dame Michael , Zora Djuric , Smith William , Max Wicha , Brenner Dean
Published: May 2015

Rationale: Dietary PUFA's alter the expression and secretion of a variety of adipokines. Diets rich in omega-6 fatty acids shift the leptin:adiponectin ratio in favor of leptin, whereas diets rich in omega-3 fatty acids have the opposite effect. We tested the hypothesis that different ratios of adipose-derived factors differentially drive or inhibit normal mammary stem cell self-renewal.

Methods: F344 rats were fed an obesogenic diet supplemented with arachidonic acid (AA) and eicosapentanoic acid (EPA) in either a 1:0 or 1:0.4 ratio. At 20 weeks, the animals were sacrificed and the visceral adipose explants were maintained in culture for up to 10 days. Conditioned media was collected at 48 hours. Adipokine concentrations were measured with the Proteome Profiler Adipokine Array and normalized to a leptin ELISA. Primary human mammary epithelial cells and the MCF10A breast cell line were expanded in a non-adherent sphere-forming assay as a measure of stem cell self-renewal. Cell suspensions were exposed to adipose conditioned media (1 mg/ml total protein concentration). After 10 days, spheres were counted and sphere size was measured using ImageJ.

Results: Adipose explants were maintained in culture for up to 10 days with >99% viability. The secretome from the rats fed omega-6 PUFA's was enriched for leptin, resistin, serpin E1, and lipocalin-2. The omega-3 rich diet led to enrichment for adiponectin, pentraxin, and RBP4 in the adipose secretome. The adipose conditioned media from the omega-6 fed rats increased mammary stem cell self-renewal by 190%, whereas that from the rats fed omega-3 PUFA's attenuated this effect.

Conclusion: The n3:n6 dietary ratio promotes a shift in the ratios of multiple secreted adipokines. These ratios have a differential effect on mammary stem cell self-renewal. A diet rich in omega-6 PUFA's leads to increased stem cell self-renewal, whereas a diet supplemented with EPA attenuated this effect. Further studies will clarify the role of specific adipokines on mammary stem cell self-renewal.

Selected adipokine levels in adipose conditioned mediaOmega-3 rich dietOmega-6 rich dietn6:n3 dietary ratio1:0.41:0.0Adiponectin1864.7778.3Leptin181.2208.7Lipocalin-244.1323.1Pentraxin-277.00.8Pentraxin-319.29.3Pref-122.98.8Resistin737.01372.3Serpin E125.877.4RBP41136.0318.1

Citation Format: Ray Esper, Michael Dame, Djuric Zora, William Smith, Wicha Max, Dean Brenner. Dietary PUFA's alter the visceral adipose secretome, which modulates mammary stem cell self-renewal. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 242. doi:10.1158/1538-7445.AM2014-242
Abstract 2420: Ribonucleotide reductase promotes genomic instability and tumor heterogeneity via increasing dUTP misincorporation into DNA
Authors: Chang Zee-Fen
Published: May 2015

Regulation of enzymes in nucleotide synthesis plays a critical role in DNA replication and repair. Ribonucleotide reductase (RNR), which is composed of RRM1/ RRM2A or RRM2B, is the key enzyme that controls the de novo synthesis of all four dNTPs, and the expression of RRM2A subunit is cell cycle-dependent. It has been well established that the level of RRM2A is often elevated in malignant tumor cells and RRM2A transgenic mice develop lung cancer. The molecular mechanism of RRM2A in cancer promotion is still unknown. In this study, we showed that constitutive elevation of RRM2A promotes replication stress, chromosome instability and cellular transformation, all of which are prevented by co-expression of dUTPase that degrades dUTP. Knockdown of dUTPase in cancer cells containing high levels of RRM2A and dUTPase also increased replication stress. Immunohistochemistry staining of surgical tumors from colorectal cancer (CRC) patients showed that high RRM2A/low dUTPase correlates with poorer overall survival, suggesting the opportunity of using RRM2/dUTPase as a prognostic indicator. We proposed that the cellular context of high RRM2/low dUTPase causes chromatin instability to drive tumor evolution, resulting in poor survival in patients. Our findings also imply that tumor cells with elevation of RRM2A are vulnerable to inhibitors against dTTP synthesis in conjunction with DNA damage agent in therapy.

Citation Format: Zee-Fen Chang. Ribonucleotide reductase promotes genomic instability and tumor heterogeneity via increasing dUTP misincorporation into DNA. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2420. doi:10.1158/1538-7445.AM2014-2420
Abstract 2426: Exacerbation of oxidative DNA damage as a synthetic lethal approach in mismatch repair deficient tumors
Authors: Fontebasso Yari , Hewish Madeleine , Lord Chris J. , Ashworth Alan
Published: May 2015

The post-replicative DNA mismatch repair (MMR) pathway plays a key role in the maintenance of genomic stability. MMR corrects mismatches within the DNA helix that arise due to errors in nucleotide incorporation during either semi-conservative replication or synthesis associated with DNA repair. The importance of this system is testified by the fact that mutations affecting components of this repair pathway cause a “mutator” phenotype and predisposition to a wide variety of malignancies including colorectal, gastric, endometrial, and urothelial cancers. For example, the hereditable disorder Lynch syndrome (hereditary nonpolyposis colon cancer) is associated with germline loss of function mutations in the MMR tumor suppressor genes MLH1, MSH2, PMS2, and MSH6. In addition to this familial syndrome, MMR deficiency is present in 15% to 17% of all colorectal cancer cases.

Synthetic lethal relationships have been identified that involve specific MMR genes, suggesting that synthetic lethal treatments could be applied to MMR deficient tumors. Previously, efforts to functionally identify genetic DNA mismatch repair synthetic lethalities and potential therapeutic targets have focused on the analysis of either candidate genes or relatively small gene subsets such as protein kinases. In order to define additional SL targets in MMR-deficient genetic backgrounds, we describe here the application of an shRNA screen in matched isogenic dMMR/pMMR (MMR-deficient/MMR-proficient) model cell lines. Using this screening approach, we identify new synthetic lethal interactions between MMR deficiency and components of the base excision repair and of the nucleotide sanitization systems, which restrict the extent of oxidative DNA damage. We propose that inhibition of the processes that minimize the oxidized DNA load could constitute a promising therapeutic approach in mismatch repair deficient cancers.

Citation Format: Yari Fontebasso, Madeleine Hewish, Chris J. Lord, Alan Ashworth. Exacerbation of oxidative DNA damage as a synthetic lethal approach in mismatch repair deficient tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2426. doi:10.1158/1538-7445.AM2014-2426
Abstract 2437: PKA/AMPK signaling is a mediator of the anti-proliferative effect of adiponectin on multiple myeloma cells
Authors: Medina Edward Antonio , Polusani Srikanth R. , Oberheu Kelli , Oyajobi Babatunde
Published: May 2015

Obesity increases the risk of developing multiple myeloma (MM). Adiponectin is a cytokine produced primarily by adipocytes, but paradoxically decreased in obesity, that has been implicated in progression of multiple myeloma. Herein, we evaluated the effects of prolonged exposure to adiponectin on the survival of MM cells as well as putative signaling mechanisms. We found that adiponectin activates PKA, which leads to decreased AKT activity and increased AMPK activation. AMPK, in turn, induces cell cycle arrest and apoptosis. Adiponectin-induced apoptosis may be mediated, at least in part, by the PKA/AMPK-dependent decline in the expression of the enzyme acetyl-CoA-carboxylase (ACC), which is critical to lipogenesis and thus cell survival. Importantly, supplementation with palmitic acid, the preliminary end product of fatty acid synthesis, rescues MM cells from adiponectin-induced apoptosis. Furthermore, 5-(tetradecyloxy)-2-furan carboxylic acid (TOFA), an ACC inhibitor, exhibited potent anti-proliferative effects on MM cells that could also be inhibited by fatty acid supplementation. Thus, the effect of adiponectin to reduce survival of MM cells appear to be mediated via its ability to suppress lipogenesis. Our findings suggest that activators of the PKA/AMPK pathway, or inhibitors of ACC, may be useful adjuvants in the treatment of MM. Moreover, the anti-myeloma effect of adiponectin supports the concept that decreased circulating levels of the adipokine, as occurs in obesity, promotes MM tumor progression.

Citation Format: Edward Antonio Medina, Srikanth R. Polusani, Kelli Oberheu, Babatunde Oyajobi. PKA/AMPK signaling is a mediator of the anti-proliferative effect of adiponectin on multiple myeloma cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2437. doi:10.1158/1538-7445.AM2014-2437
Abstract 245: Consumption of a high-fat diet abrogates inhibitory effects of selenium on spontaneous metastasis of Lewis lung carcinoma in mice
Authors: Yan Lin
Published: May 2015

The present study investigated the effects of dietary supplementation with selenium (Se) on spontaneous metastasis of Lewis lung carcinoma (LLC) in mice fed a high-fat diet using a 2x2 design. Three-week-old male C57BL/6 mice were fed the AIN93G diet or that diet modified with 45% calories from fat supplemented with or without 2.5 mg Se/4029 kcal as methylseleninic acid for 6 weeks, at which time they were injected subcutaneously with 2.5x105 LLC cells. The resulting primary tumor was removed surgically 10 days later, and the experiment was terminated after an additional 10 days. Consumption of the high-fat diet significantly increased the body fat mass, regardless of Se supplementation, compared to the AIN93G diet (p<0.01). The high-fat diet increased lung metastases by 17% compared to the AIN93G diet (p<0.01). Selenium supplementation reduced the metastases by 11% compared to non-supplemented controls (p<0.05); however, the reduction was greater in AIN93G-fed mice (18%) than in mice fed the high-fat diet (5%). Supplemental Se reduced plasma concentrations of proteolytic proteases (uPA, p<0.01; MMP-9, p<0.05) and angiogenic factors (VEGF, p<0.01; TIMP-1, p<0.01) compared to non-supplemented controls. The high-fat diet significantly increased plasma concentrations of adipokines PAI-1, MCP-1, TNF-α and leptin regardless of the level of dietary Se; however, supplemental Se reduced plasma PAI-1 (p≤0.05) and MCP-1 (p≤0.05) in AIN93G-fed mice. These results demonstrate that consumption of a high-fat diet abrogated the anti-metastatic effects of Se, and that such an abrogation may involve adipose-derived inflammatory cytokines.

Citation Format: Lin Yan. Consumption of a high-fat diet abrogates inhibitory effects of selenium on spontaneous metastasis of Lewis lung carcinoma in mice. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 245. doi:10.1158/1538-7445.AM2014-245
Abstract 2454: Ampk and hdac pathways and regulation rassf1c gene expression
Authors: Amaar Yousef G. , Reeves Mark E. , Firek Matthew
Published: May 2015

Introduction: We have previously shown that RASSF1C promotes cell proliferation, migration and attenuates apoptosis in cancer cells. We have also shown that RASSF1C is significantly up-regulated in breast and lung cancer tissues compared to normal tissues. Together these demonstrate that RASSF1C, is not a tumor suppressor like RASSF1A, but instead appears to function as an oncoprotein.

Currently nothing is known about the upstream signaling cascades involved in regulating RASSF1C gene expression and thus we performed transcriptome PCR array study to identify chemicals inhibitors that modulate(s) RASSF1C expression.

Method: A transcriptome PCR array was obtained from Qiagen. It consisted of cDNA from breast cancer cell line MCF7 cells treated with 90 different chemical inhibitors that regulate various signaling pathways. The array was screened with RASSF1C gene specific primers. Data analysis was performed by importing the Ct values obtained into data analysis software.

Results: The PCR array screen identified several chemical reagents that appear to up-regulate and several that seem to down-regulate RASSF1C expression by ≥ 1.5 fold. The two most notable reagents are Dorsopmorhin (AMPK inhibitor) which up-regulates RASSF1C expression by 2.8 fold and Trichostatin A (HDAC inhibitor) which down regulates RASSF1C expression by 2 fold. We are in process of confirming the effect of these inhibitors in a panel of breast and lung cancer cells.

Conclusion: The findings are novel and suggest that inhibition of AMPK pathway induces RASSF1C expression while inhibition of HDAC pathway suppresses RASSF1C gene expression. We should note that Trichostatin A has been shown to activate AMPK, inhibits HDAC, arrests cell growth, and induces apoptosis in human cancers. Our findings provide a potential mechanism for regulating RASSF1C gene expression through the modulation of AMPK and HDAC pathways.

Citation Format: Yousef G. Amaar, Mark E. Reeves, Matthew Firek. Ampk and hdac pathways and regulation rassf1c gene expression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2454. doi:10.1158/1538-7445.AM2014-2454
Abstract 2458: Automated analysis of four color PTEN deletion in prostate cancer using FISHQuant on a whole slide image
Authors: Csizmadia Annamaria , Szipocs Ferenc , Park Janet , Harris Mohammmed , Hartmayer Peter , Moss Thomas J.
Published: May 2015

Mutations within the Phosphatase and tensin homolog (PTEN) gene have been linked to the development of many cancers. PTEN is one of the most commonly lost tumor suppressors in human cancer; in fact, up to 40% of men with prostate cancer are estimated to have lost a copy of the PTEN gene at the time of diagnosis. Slides stained with CymogenDx's PTEN del-TECT FOUR COLOR probe, developed for reliably detecting PTEN deletion, are scanned using 3DHISTECH's Panoramic 250 FLASH II digital slide scanner and the resulting whole slides are subsequently analyzed using 3DHISTECH's FISHQuant software. The ability to capture fluorescent signals from 9 independent channels and up to 29 different Z layers with perfect co-localization enables the creation of multiple labelled fluorescent slides. 3DHISTECH's “Extended Focus” feature creates a single in-focus image where all the in-focus parts of each captured layer is merged into one composite image. This enables the use of automated algorithms to evaluate different FISH probes in tissue and cytology whole slide samples. The FISHQuant software segments nuclei and spots automatically and scores the resulting data. For PTEN deletion, it measures the deletion clusters for the 3 colors involved. It also uses the control to check for numerical deviation of the 10th chromosome's signals. These two measurements are run automatically and displayed on a two-dimensional “HistoPlot”. This proof of concept study, using prostate core needle biopsies, demonstrates that the PTEN deletion FISH signals captured on a digital slide and overlaid in bright field and fluorescence modes can be subsequently analyzed using automated algorithms. The ability to identify tumor heterogeneity in a prostate core biopsy (and other sample preps) thus becomes possible. “Extended focus” and the patent pending PTEN del-TECT FOUR COLOR probe limits truncations artifact and makes a huge difference as sections get thinner due to other tests needing to be performed. Having access to the whole slide for analysis provides enough cells of interest.

Citation Format: Annamaria Csizmadia, Ferenc Szipocs, Janet Park, Mohammmed Harris, Peter Hartmayer, Thomas J. Moss. Automated analysis of four color PTEN deletion in prostate cancer using FISHQuant on a whole slide image. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2458. doi:10.1158/1538-7445.AM2014-2458
Abstract 2459: The oncogenic activity of MDMx is associated with physical interaction and suppression of retinoblastoma protein
Authors: Xiao Zhi-Xiong , Zhang Haibo , Zhang Yujun
Published: May 2015

The aim of this study is to investigate the role of MDMx (or MDM4) on the regulation of retinoblastoma protein (Rb). Inactivation of Rb plays a critical role in the development of human malignancies. We have previously shown that MDM2, an ubiquitin E3 ligase and a major negative regulator of p53, binds to and promotes proteasome-mediated degradation of Rb independent of p53. MDMX, a homolog of MDM2, also binds to and inhibits p53 transactivation activity, yet it does not possess an ubiquitin E3 ligase activity. In this study, we show that MDMX binds to and promotes proteasome-mediated degradation of Rb in a MDM2-dependent manner. The C-terminal Ring domain of MDMX binds to the Rb C-pocket and enhances MDM2-Rb interaction. Knockdown of MDMX induces Rb protein accumulation, cell cycle G1 arrest, and premature cellular senescence, which can be reverted by simultaneous knockdown of Rb. Furthermore, ablation of MDMX significantly suppresses tumor growth, concomitant with Rb accumulation, in an animal xenograft model. Together, these results demonstrate that MDMX possesses oncogenic activity associated with suppression of Rb and suggest that both MDM2 and MDMX can be targets for cancer therapy.

Note: This abstract was not presented at the meeting.

Citation Format: Zhi-Xiong Xiao, Haibo Zhang, Yujun Zhang. The oncogenic activity of MDMx is associated with physical interaction and suppression of retinoblastoma protein. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2459. doi:10.1158/1538-7445.AM2014-2459
Abstract 2462: Coiled-coil domain 68 (CCDC68) plays a tumor suppressive role in pancreatic adenocarcinoma
Authors: Radulovich Nikolina , Leung Lisa , Ibrahimov Emin , Navab Roya , Sakashita Shingo , Lockwood William , Thu Kelsie , Fedeshyn Yaroslav , Moffat Jason , Lam Wan , Tsao Ming
Published: May 2015

Pancreatic ductal adenocarcinoma (PDAC) is driven by somatic alterations. Recent studies revealed that most genetic alterations are represented by rare mutations in many genes that are either drivers or passengers of PDAC. Using integrative genomics and functional screenings we identified coiled-coil domain 68 (CCDC68) as a putative tumor suppressor (TSG) in PDAC. Combination of Q-RT-PCR and FISH analysis revealed CCDC68 allelic losses in 50% of PDAC cell lines and 44% of primary patient xenografts. 21% of PDAC cell lines and primary patient xenografts also showed decreased levels of CCDC68 mRNA and protein. We also report the discovery of an unstable novel alternate splice variant (SNP rs. 1344011) of CCDC68 expressed by 30% of PDAC patients. Overexpression of full length CCDC68 (CCDC68wt) in PANC-1 and Hs.766T cell lines significantly decreased their cell proliferation, anchorage-independent growth and in vivo tumorigenicity in scid mice. However, the overexpression of novel transcript CCDC68 variant did not affect the cell proliferation or tumorigenicity potential of PANC-1 cells. Finally, we identified that CCDC68 negatively regulates MAPK signalling independently of KRAS status. In conclusion, CCDC68 loss-of-function through copy loss, mRNA downregulation and truncated protein expression suppresses the tumorigenicity of PDAC cell lines suggesting that CCDC68 is a candidate novel TSG in PDAC.

Citation Format: Nikolina Radulovich, Lisa Leung, Emin Ibrahimov, Roya Navab, Shingo Sakashita, William Lockwood, Kelsie Thu, Yaroslav Fedeshyn, Jason Moffat, Wan Lam, Ming Tsao. Coiled-coil domain 68 (CCDC68) plays a tumor suppressive role in pancreatic adenocarcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2462. doi:10.1158/1538-7445.AM2014-2462
Abstract 2464: Angiomotin plays a tumor suppressor role by sequestering oncogenic YAP/TAZ proteins and decreasing amphiregulin secretion in lung cancer
Authors: Kuo Po-Lin , Hung Jen-Yu , Hsu Ya-Ling
Published: May 2015

Lung cancer is the leading cause of cancer death worldwide, with metastasis underlying the majority of lung cancer-related deaths. Angiomotin (AMOT), a scaffold protein, has been shown to interact with oncogenic YAP/TAZ proteins, suggesting its potential role in tumor suppression. However, the functional role of AMOT in lung cancer remains unknown. We observed that the expression of AMOT was significantly decreased in clinical lung cancer specimens. Knockdown of AMOT in a low metastatic CL1-0 lung cancer cell line initiated cancer proliferation, migration, invasion and epithelial-mesenchymal transition. The trigger of cancer progression caused by AMOT loss was transduced by decreasing cytoplasmic sequestration and increasing nuclear translocation of oncogenic co-activators YAP/TAZ, resulting in an increased expression of the growth factor amphiregulin. The tumor promotion by AMOT knockdown was reversed when YAP/TAZ were absent or anti-amphiregulin antibodies were present. Further, AMOT knockdown increased the spread of Lewis lung carcinoma in vivo. Our findings suggest that AMOT is a crucial suppressor of lung cancer metastasis. These observations highlight the critical role of AMOT as a tumor suppressor, and the potential of AMOT as a prognostic biomarker and potential therapy target for lung cancer.

Note: This abstract was not presented at the meeting.

Citation Format: Po-Lin Kuo, Jen-Yu Hung, Ya-Ling Hsu. Angiomotin plays a tumor suppressor role by sequestering oncogenic YAP/TAZ proteins and decreasing amphiregulin secretion in lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2464. doi:10.1158/1538-7445.AM2014-2464
Abstract 2467: Connexin43 reduces melanoma growth within a keratinocyte microenvironment and during tumorigenesis in vivo
Authors: Shao Qing C. , Ableser Mark Jake , Penuela Silvia , Laird Dale W.
Published: May 2015

Gap junctions formed from connexins (Cx) have been reported to regulate proliferation, differentiation and apoptosis through formation of intercellular channels between opposing cells. Interestingly, connexins have been identified as tumor suppressors and enhancers, a distinction dependent on the type and stage of disease. In melanomas, the role of connexins during cancer onset and progression remains unclear. In the current study, we hypothesize that expression of Cx26 and Cx43 in BL6 mouse melanomas will increase gap junctional intercellular communication (GJIC) thereby decrease their tumorigenic properties. Western blots, immunofluorescence and dye coupling studies revealed that BL6 cells express low levels of Cx26 , Cx43 and are poorly coupled. When cells were engineered to express Cx26 or Cx43, dye transfer studies revealed that both Cx26- and Cx43-expressing cells were highly coupled. Interestingly, when these connexin-rich melanomas were co-cultured with Cx43-rich keratinocytes, melanoma microtumors remained functionally isolated and failed to couple to surrounding keratinocytes. Growth curve studies revealed that exogenous Cx43, but not Cx26, significantly reduced total cell numbers; that was primarily due to reduced cell proliferation. Furthermore, Cx43 expression significantly reduced anchorage-independent growth compared to controls, however, migration remained unaffected. Additionally, Cx43-expressing melanoma cells displayed a reduction in growth when co-cultured with keratinocytes, despite a lack of heterocellular GJIC. Finally, when grown in in vivo avian embryo model, primary tumor weight was reduced in tumor cells expressing Cx43 , but not in Cx26-expressing melanomas , as compared to controls. Overall, our results would suggest that Cx43 is a potent tumor suppressor in melanomas. Supported by the Canadian Institute of Health Research to DWL.

Citation Format: Qing C. Shao, Mark Jake Ableser, Silvia Penuela, Dale W. Laird. Connexin43 reduces melanoma growth within a keratinocyte microenvironment and during tumorigenesis in vivo. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2467. doi:10.1158/1538-7445.AM2014-2467
Abstract 2475: Zpo2 promotes aggressive breast cancer development through downregulation of GATA3
Authors: Shahi Payam , Slorach Euan M. , Chou Jonathan , Lawson Devon , Yu Ying , Werb Zena
Published: May 2015

Metastatic breast cancer is the major cause of poor prognosis and death in women. The transcription factor GATA3 is a key mediator of luminal cell fate determination and homeostasis in adult mammary gland. Loss of GATA3 has been associated with development of metastatic mammary tumors. Here we describe a novel role for transcriptional repressor, Zeppo2 (zinc finger elbow-related proline domain protein2; Zpo2) (ZNF503) in promoting metastatic breast cancer through modulation of GATA3. We demonstrate that Zpo2, through its interaction with repressor of GATA (ROG) is capable of modulating GATA3 transcription in mammary epithelial cells. Overexpression of Zpo2 results in upregulation of EMT associated genes, decreased cell-cell adhesion and increased cellular invasiveness in 3D cultures. Additionally, overexpression of Zpo2 in mammary tumor cells results in enhancement of tumor growth and increased metastasis in orthotopic transplant models. Furthermore, to investigate the importance of Zpo2 in human breast cancer metastasis, we utilized a series of new patient-derived xenograft (PDX) models (primary human xenograft models). The PDX models ranged from poorly metastatic to highly metastatic as was observed in the (affected) original patients. Interestingly, higher Zpo2 expression levels correlate with more aggressive xenograft tumor models. Collectively, our data suggests that Zpo2 plays a potentially significant role in initiating aggressive breast cancer by modulating GATA3 in mammary epithelial cells.

Citation Format: Payam Shahi, Euan M. Slorach, Jonathan Chou, Devon Lawson, Ying Yu, Zena Werb. Zpo2 promotes aggressive breast cancer development through downregulation of GATA3. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2475. doi:10.1158/1538-7445.AM2014-2475
Abstract 2480: Ubiquitination of p53 by Trim39
Authors: Zhang Liguo , Chen Chen , Huang Naijia , Tang Wanli , Kornbluth Sally
Published: May 2015

Trim39 is a RING domain-containing E3 ubiquitin ligase able to directly inhibit the anaphase promoting complex (APC/C). Through analysis of Trim39 function in p53-positive and p53-negative cells we have found, surprisingly, that p53-positive cells lacking Trim39 could not traverse the G1/S transition. This effect did not result from dis-inhibition of the APC/C. Moreover, while Trim39 loss inhibited etoposide-induced apoptosis in p53-negative cells, apoptosis was enhanced by Trim39 knock-down in p53-positive cells. Furthermore, we show here that the Trim39 can directly bind and ubiquitylate p53 in vitro and in vivo, leading to p53 degradation. Depletion of Trim39 significantly increased p53 protein levels and cell growth retardation in multiple cell lines. We found that the relative importance of Trim39 and the well-characterized p53-directed E3 ligase, MDM2, varied between cell types. In cells that were relatively insensitive to the MDM2 inhibitor, nutlin-3a, apoptosis could be markedly enhanced by siRNA directed against Trim39. As such, Trim39 may serve as a potential therapeutic target in tumors with wild type p53 when MDM2 inhibition is insufficient to elevate p53 levels and apoptosis.

Citation Format: Liguo Zhang, Chen Chen, Naijia Huang, Wanli Tang, Sally Kornbluth. Ubiquitination of p53 by Trim39. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2480. doi:10.1158/1538-7445.AM2014-2480
Abstract 2482A: ARHI (DIRAS3) downregulates Ras-MAPK signaling and inhibits transformation of NIH3T3 cells through direct interaction with GTP-bound Ras
Authors: Sutton Margie N.
Published: May 2015

Somatic mutation of Ras was the first genetic alteration documented to drive human oncogenesis. Activating mutations of H-Ras, N-Ras, or K-Ras are associated with 30% of all human cancers. Characterization of wild type Ras proteins has identified their essential role in intra-cellular signaling networks controlling cell proliferation, differentiation and survival. Despite intensive efforts, effective targeted therapy directed against mutated Ras has remained elusive. Regulation of Ras function has been studied in depth, but relatively little attention has been paid to physiological inhibitors of Ras. Our group has previously reported that the maternally imprinted Ras-related tumor suppressor gene ARHI (DIRAS3) is downregulated in more than 60% of ovarian cancers and several other tumor types. Re-expression of ARHI at physiologic levels can inhibit ovarian cancer cell proliferation, block migration, induce autophagy, mediate tumor dormancy and block both PI3K and Ras-MAPK signaling. ARHI shares 50-60% homology with the H- and K-Ras genes, differing primarily in a 34-amino acid N-terminal extension that is required for most of ARHI's functions. The mechanism by which ARHI regulates Ras-MAPK signaling is not fully understood. Here, we show that re-expression of ARHI in NIH3T3 murine fibroblasts inhibits cell proliferation, suppresses clonogenic growth and blocks cell transformation by constitutively active mutant H-Ras. Addition of the N-terminal extension of ARHI to mutant H-Ras also prevents malignant transformation. ARHI interacts directly with mutated Ras on co-immunoprecipitation and can inhibit GTP-bound Ras activity following overexpression of the constitutively active construct. We hypothesize that inhibition of the Ras-MAPK signaling pathways following re-expression of ARHI (DIRAS3) may be, in part, the effect of a direct interaction between GTP-bound Ras and ARHI, resulting in the restoration of intrinsic Ras GTPase activity. Our results suggest that ARHI (DIRAS3) may serve as a novel inhibitor of activated Ras, and re-expression of ARHI in tumors exhibiting an oncogenic mutation of Ras might permit more specifically targeted therapy.

Citation Format: Margie N. Sutton. ARHI (DIRAS3) downregulates Ras-MAPK signaling and inhibits transformation of NIH3T3 cells through direct interaction with GTP-bound Ras. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2482A. doi:10.1158/1538-7445.AM2014-2482A
Abstract 2493: Molecular biomarkers for cancer biospecimen integrity
Authors: Agrawal Lokesh , Vaught Jim , Moore Helen
Published: May 2015

Successful implementation of personalized health care requires the availability of biospecimens of sufficient quality to ensure accurate downstream test results, which will in turn determine optimal patient management. Cancer biospecimens of known quality with appropriate clinical annotation are also critical to the process of biomarker discovery and validation that lead to the development of clinical tests. Due to widely varying methods of biospecimen collection, processing, and storage, numerous pre-analytical factors can affect human biospecimen integrity for biomarker research. Such pre-analytical variability can affect the integrity of research biospecimens including plasma/serum and fixed cancer tissues used for biomarker analysis. The National Cancer Institute (NCI) Biorepositories and Biospecimen Research Branch (BBRB) was established to address the scientific and policy issues that affect the procurement of and access to high quality specimens for research. The NCI Biospecimen Research Network (BRN) was established to sponsor and conduct research on biospecimen pre-analytical factors and their affect on molecular integrity, to support the development and adoption of evidence-based standardized biospecimen handling procedures. In this presentation we will summarize BRN-supported studies to identify biomarkers that may be used as sentinel markers of plasma stability using mass spectroscopy proteomics and analysis of circulating miRNA, and the development of intrinsic controls for FFPE tissues. In one of the projects, identification of protein biomarkers using mass-spectrometry and Illumina arrays in plasma led to the development of a biomarker panel for serum/plasma integrity; these biomarkers are currently being validated. A second project studied the effects of pre-analytical variables on circulating miRNA and identified and validated new and improved housekeeping miRNA, as well as novel biomarkers associated with breast cancer. In another study, a series of biomarkers have been validated by construction of tissue microarrays from 93 breast cancer specimens with known time to fixation delays. A tissue quality index (TQI) model was generated to predict the time to fixation and tissue quality by measuring a subset of biomarker proteins in breast cancer tissues using AQUA technology. The progressive efforts taken by BRN investigator-led projects to identify and validate biomarkers for sample integrity will be incorporated into efforts to develop new biospecimen evidence-based practices for clinical biomarker research.

Citation Format: Lokesh Agrawal, Jim Vaught, Helen Moore. Molecular biomarkers for cancer biospecimen integrity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2493. doi:10.1158/1538-7445.AM2014-2493
Abstract 2498: Detection of S-nitrosylated heat shock protein 90 in renal cell cancer
Authors: Hongo Fumiya , Ueda Takashi , Ito-Ueda Saya , Oishi Masakatsu , Nakamura Terukazu , Naya Yoshio , Miki Tsuneharu
Published: May 2015

Purpose: Nitric oxide (NO) is a versatile signaling molecule. Its roles in a variety of physiological functions in mammals are beginning to be understood. It has been demonstrated that NO up-regulates the expression of the Fas receptor on human tumor cells via specific inactivation of transcription repressor YY1 DNA binding activity to the silencer region of the Fas promoter (Hermes G., et al. 2001). We also showed that inactivation of YY1 via protein S-nitrosylated by NO in prostatic cancer could be identified (Hongo F., et al. 2005). This post-translational modification, known as S-nitrosylation, has emerged as a highly conserved and spatiotemporally specific signaling mechanism. The objective of our study was to examine the effect of NO donor treatment on renal cell cancer.

Methods: We examined the effect of the NO donor DETA-NONOate (100-500uM) on the following renal cell cancer cell lines; CAKI-1, NC65, and ACHN. Anti-S-nitroso-Cysteine antibody in the rabbit (SIGMA-ALDRICH) was applied as a primary antibody for immunohistochemistry. The biotin-switch technique was applied to detect specific S-nitrosylated proteins. Whole cell lysates were extracted from ACHN cells employed with DETA-NONOate. Using an S-nitrosylated detection kit (Cayman Chemical Company), S-NO was replaced by Biotin. Cell lysates were incubated at 4 oC with 30 μL of Dynabeads M-280 Streptavidin (Invitrogen) for 3 hrs. After five washes with wash buffer (S-Nitrosylation Wash buffer), the protein complex was eluted from anti-biotin dynabeads by incubation with 1x volume of elution buffer (0.1M glycine (pH2.0)) twice for 15 min. Each sample was added to an SDS-polyacrylamide gel for mass spectrometry.

Results: Significant up-regulation of S-nitrosylated proteins employed with DETA-NONOate was observed by immunohistochemistry. Specific S-nitrosylated proteins in ACHN cells were detected by the biotin-switch technique and included HSP90 (heat shock protein 90), GRP78 (HSP70 family), HSP70, and pyruvate kinase M2. In ACNH cells, Significant up regulation of S-nitrosylated HSP90 by western blot.

Conclusions: Our data showed that NO treatment significantly increased S-nitrosylation of several proteins. S-nitrosylation should be considered as one of the mechanisms by which NO acts on cancer cells.

Citation Format: Fumiya Hongo, Takashi Ueda, Saya Ito-Ueda, Masakatsu Oishi, Terukazu Nakamura, Yoshio Naya, Tsuneharu Miki. Detection of S-nitrosylated heat shock protein 90 in renal cell cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2498. doi:10.1158/1538-7445.AM2014-2498
Abstract 2503: Differential expression of cytoskeletal proteins in acute lymphoblastic leukemia in accordance with initial response to chemotherapy
Authors: Kang Kee R. , Park Eun Sil
Published: May 2015

Precursor B lymphoblastic leukemia is a B-cell lineage neoplasm of lymphoblasts. The initial response to induction-chemotherapy is the most important prognostic factor in acute lymphoblastic leukemia (ALL) patients. Patients with rapid-early-response (RER, below 5% of blast in bone marrow at day-7 after induction-chemotherapy) have better event-free survival compared to slow-early-responders (SER, above 25% of blast). The drug sensitivity/resistance of leukemic blasts is mainly determined by specific genetic abnormalities, however, in more than 20% of ALL, the abnormalities remain unclassified, and the treatment outcome persist variable. This study aimed to find plausible biomarkers of drug sensitivity/resistance in ALL using proteomic analysis. We identified 28 up-regulated and 25 down-regulated proteins in SER_as compared to RER_by MALDI-TOF and mass spectrometry. Of these, 14-3-3 isoforms and cytoskeletal proteins were differentially expressed. Alpha tubulin and 14-3-3 gamma, down-regulated in RER were further verified by Western blot analysis. In further studies, the roles of these proteins in drug sensitivity/resistance are undergoing to elucidate as a potential biomarker for ALL therapy.

Citation Format: Kee R. Kang, Eun Sil Park. Differential expression of cytoskeletal proteins in acute lymphoblastic leukemia in accordance with initial response to chemotherapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2503. doi:10.1158/1538-7445.AM2014-2503