bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2369: Molecular biological characterization of newly established epithelial ovarian cancer (EOC) cell lines : The cyclin-dependent kinase 1 pathway
Authors: yang wookyeom , Cho hanbyoul , Shin hayeon , Lee eunju , Kwon hyunja , Kim sol , Kim jaehoon
Published: May 2015

Background

Gene expression pattern analysis comparing normal cells with cancer cells are performed by many competitors in the worldwide lab. Cancer has many different species in itself due to its individual differences, variations and heterogeneity. Therefore, it is necessary to spend more time in gene expression pattern analysis using microarray or whole genome sequencing. The purpose of this study was to identify genes and proteins that are highly differentially expressed in newly established epithelial ovarian cancer (EOC) cell lines, and to use this knowledge for the development of novel diagnostic and prognostic markers for EOC. We further performed the validation studies of several candidate genes.

Results

Comparison of gene expression patterns using microarray analysis enabled us to identify 84 genes that were commonly up-regulated and 132 genes that were down-regulated in cancer cell lines (up; > 2-fold, down; < 3-fold, P < 0.05). In 2-DE and MALDI-TOF/PMF, 31 up-regulated spots that had at least two-fold differences between the 3 EOC cell lines and HOSE cells used as controls were observed. 15 up-regulated genes in cell cycle related genes and 9 down-regulated genes in apoptosis related confirmed gene expression using quantitative RT-PCR. Pathway analysis was validated in cell cycle related genes in six EOC cell lines.

Conclusions

The data shows the aberrant expression of several cell cycle related genes in EOC and that Cyclin-dependent kinase 1 (CDK1) pathway may play a significant role in ovarian carcinogenesis, implying that CDK1 pathway could be used as a novel therapeutic target for ovarian cancer treatment.

Citation Format: wookyeom yang, hanbyoul Cho, hayeon Shin, eunju Lee, hyunja Kwon, sol Kim, jaehoon Kim. Molecular biological characterization of newly established epithelial ovarian cancer (EOC) cell lines : The cyclin-dependent kinase 1 pathway. [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 2369. doi:10.1158/1538-7445.AM2014-2369
Abstract 2372: Detecting driver genes based on tumor whole-exome sequencing studies
Authors: Hua Xing , Landi Teresa Maria , Shi Jianxin
Published: May 2015

Driver somatic mutations confer a selective growth advantage to tumors while passenger mutations do not. Genes carrying driver mutations are detected in whole-exome sequencing (WES) studies if their non-synonymous mutations are significantly more frequent than their silent mutations, adjusting for the heterogeneous mutation rates across genomic locations, subjects and mutation types. Typical tumor WES studies (∼100-300 tumor samples) lack statistical power, and thus can identify only small numbers of candidate driver genes and possibly miss the well-established ones. The best algorithm to detect driver genes to date is MutSigCV. For each target gene, MutSigCV identifies its “bagel”, a set of genes predicted to have similar background mutation rates to the target gene and use the “bagels” to estimate the background mutation rate. Based on the identified “bagels”, we developed a novel statistical method implementing a series of algorithms to improve the statistical power while maintaining the type-I error rate. First, we developed a statistical framework for testing the selective growth advantage of each mutation type and appropriately integrating evidence of growth advantage across mutation types to achieve robust power. We then extended the algorithm to consider spatial clustering information. This substantially improves the statistical power when non-silent mutations are clustered into a few short regions or nucleotides. We applied this novel method to TCGA melanoma WES including 271 tumor samples. Without modeling spatial clustering information, over 30 candidate driver genes, including BRAF, NRAS, PTEN, TP53, PPP6C and CDKN2A were detected controlling FDR=0.01. Modeling spatial information of somatic mutations detected additional rarely mutated genes, including DISP1 (P=1.3×10-20, involved in cellular proliferation and differentiation that leads to normal development of embryonic structures), IDH1 (P=1.0×10-8, previously identified in relation to gliobalstoma and acute myeloid leukemia), MAP2K1 (P=5×10-18, a member of the mitogen-activated protein kinases that also include BRAF, MEK and other important players in melanoma), INTS8 (P=7.7×10-15, an integrator of small nuclear RNA processing), and STK19 (P=1.2×10-10, encoding a serine/threonine kinase, previously identified in melanoma). Finally, we developed methods to simultaneously analyze multiple cancers to improve the power for genes mutated in more than one cancer type, which will be illustrated by analyzing 12 major cancers in TCGA. Our method will provide an important tool for improving driver gene mutation detection in cancer.

Citation Format: Xing Hua, Teresa Maria Landi, Jianxin Shi. Detecting driver genes based on tumor whole-exome sequencing studies. [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 2372. doi:10.1158/1538-7445.AM2014-2372
Abstract 2384: Time-lapse imaging of response to DNA damage occuring during mitosis
Authors: Miwa Shinji , Yano Shuya , Yamamoto Mako , Hiroshima Yukihiko , Uehara Fuminari , Matsumoto Yasunori , Kimura Hiroaki , Hayashi Katsuhiro , Efimova Elena V. , Tsuchiya Hiroyuki , Hoffman Robert M.
Published: May 2015

The DNA damage response protein 53BP1 forming foci when double-stranded breaks occur in DNA. In this study, the response to DNA damage during mitosis was visualized using fluorescent protein-based real-time imaging of 53BP1 linked to GFP (GFP-53BP1) focus formation by the fusion protein in the MiaPaCa2 Tet-On Advanced cell line. The MiaPaCa2Tet-On GFP-53BP1 cells were cultured in 35 mm dishes for 48 h. 53BP1 foci were observed every 30 minutes with the FluoView FV1000 confocal laser microscope (Olympus Corp., Tokyo, Japan). The increased expression of GFP-53BP1 was observed during mitosis in MiaPaCa2Tet-On GFP-53BP1 cells without any treatment Mitotic changes were observed 1.09 ± 0.10 times in each cell during over 24 h. Apoptotic changes were observed in 7.3 ± 3.3% of the cells during the 24 h. During the time-lapse imaging, focus formation of GFP-53BP1 was observed in 11.4 ± 2.1% of the mitotic cells during over 24 h. Non-mitotic cells did not have an increase in GFP-53BP1 focus formation. This study indicates that DNA strand breaks occur during mitosis and can be repaired, at least to some extent.

Citation Format: Shinji Miwa, Shuya Yano, Mako Yamamoto, Yukihiko Hiroshima, Fuminari Uehara, Yasunori Matsumoto, Hiroaki Kimura, Katsuhiro Hayashi, Elena V. Efimova, Hiroyuki Tsuchiya, Robert M. Hoffman. Time-lapse imaging of response to DNA damage occuring during mitosis. [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 2384. doi:10.1158/1538-7445.AM2014-2384
Abstract 2396: Widespread occurrence of extrachromosomal microDNAs in normal and cancerous vertebrate tissues and cell lines
Authors: Dillon Laura , Kumar Pankaj , Shibata Yoshiyuki , Dutta Anindya
Published: May 2015

MicroDNAs are small (<400 base pairs in length) single- or double-stranded extrachromosomal circular DNAs that are derived by excision from tens of thousands of sites within the mammalian genome. Thus far, microDNAs have been detected in several mouse tissues, mouse and human cell lines, and both normal and malignant cells. Here, we examine the properties of microDNAs across ten tissue types (brain, heart, lung, liver, thymus, spleen, kidney, skeletal muscle, testis and sperm) from normal adult mice, chicken cell lines and human cancer cell lines. We observed microDNAs in all tissue types, originating from thousands of unique genomic loci. As was previously described, these microDNAs are small circles (100 to 400 base pairs), possess a high GC content and are enriched in genic regions. Hot spots of microDNA generation were noted across all tissue types and correlate with high gene density and GC content. Previously, we suggested that the release of microDNAs leaves behind microdeletions within the source genomic loci, resulting in somatically mosaic cells. Our results confirm this possibility by demonstrating that microDNAs are universally present across cell and tissue types in vertebrates. Interestingly, we found altered distribution patterns in the human cancer cell lines. In addition, short (2-15 bp) direct repeats that usually flank the genomic loci yielding microDNAs lead us to hypothesize that DNA damage repair pathways may be involved in microDNA generation. A survey of microDNAs in cell lines defective in proteins required for many DNA repair pathways revealed that the homologous recombination and non-homologous end joining pathways are dispensable for microDNA formation. However, deletion of two proteins, MSH3 and AID, resulted in a significant increase of microDNA originating from CpG islands. These observations combined with those in the cancer cell lines suggest that perturbation of normal cellular processes can alter the generation of microDNA.

Citation Format: Laura Dillon, Pankaj Kumar, Yoshiyuki Shibata, Anindya Dutta. Widespread occurrence of extrachromosomal microDNAs in normal and cancerous vertebrate tissues and cell lines. [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 2396. doi:10.1158/1538-7445.AM2014-2396
Abstract 2404: Cumulative dosage effect of TSGs and OGs drives aneuploidy patterns in cancer
Authors: Davoli Teresa , Xu Andrew Wei , Mengwasser Kristen E. , Sack Laura M. , Yoon John C. , Park Peter J. , Elledge Stephen J.
Published: May 2015

Since Boveri's postulates in 1914, aneuploidy has been recognized as a hallmark of human cancer. However, no comprehensive theory exists to explain the patterns of aneuploidy and its role in tumor evolution. Recurrent gains or losses of specific chromosomes have been observed in cancer, but it is not clear whether a recurrent pattern of aneuploidy exists among cancer karyotypes and whether the recurrent patterns just happen to occur more frequently or are specifically selected during tumor evolution. 

We have developed the Tumor Suppressor and Oncogene (TUSON) Explorer, a computational method for the prediction of tumor suppressor genes (TSGs) and oncogenes (OGs) through the analysis of the pattern of somatic mutations in cancer (Davoli et al., Cell, 2013). By analyzing >8200 tumor-normal pairs from more than 20 tumor types we provide statistical evidence suggesting that many more genes possess cancer driver properties than anticipated, forming a continuum of oncogenic potential. The TUSON Explorer analysis on the entire dataset has identified new potential TSGs such as RASA1, TP53BP1, USP28 and OGs such as PPP2R1A and PPP6C. The TUSON Explorer was also applied to individual tumor types, leading to the identification of new potential tissue-specific drivers.

In addition, we have integrated our prediction of TSGs and OGs with information of copy number alterations. We found that the distribution on chromosome and the potency of TSGs and OGs can predict the frequency of arm-level and chromosome-level deletions and amplification. For example, the density and potency of TSGs strongly positively and negatively correlated with the frequency of arm and chromosome deletion and amplification, respectively (r value=∼0.6, p<10-4). The opposite behavior is seen for the OG density and potency. We find that by combining the TSG, OG and essential gene density and potency in a unified score, we can predict a high fraction of the recurrent arm and chromosome deletions and amplifications (r value= ∼0.8, p<10-9). We propose that it is the decrease or increase in the gene dosage of cancer drivers and essential genes in deletion and amplification to determine the recurrent patterns of aneuploidy selected during tumor evolution. In this view the aneuploidy state of tumor cells, first recognized by Bovery exactly a century ago, represents a driving force during the evolution of human cancer.

Citation Format: Teresa Davoli, Andrew Wei Xu, Kristen E. Mengwasser, Laura M. Sack, John C. Yoon, Peter J. Park, Stephen J. Elledge. Cumulative dosage effect of TSGs and OGs drives aneuploidy patterns in 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 2404. doi:10.1158/1538-7445.AM2014-2404
Abstract 2421: Small molecule inhibitors of DNA glycosylases as potential drugs in cancer therapy
Authors: Dizdaroglu Miral , Jaruga Pawel
Published: May 2015

DNA glycosylases are required to initiate the DNA base excision repair pathway of lesions that are formed by DNA damage. Recently, several DNA glycosylases including NEIL1, OGG1 and NTH1 were identified as potential targets in combination chemotherapeutic strategies for cancer treatment. The potential therapeutic benefit for the inhibition of DNA glycosylases was validated by demonstrating synthetic lethality with drugs that are commonly used to limit DNA replication through dNTP pool depletion. Additionally, NEIL1-associated synthetic lethality has been achieved in combination with Fanconi anemia, group G. In this work, we designed experimental strategies to discover small molecule inhibitors of NEIL1, with subsequent analyses of the specificity of those molecules for other DNA glycosylases. Using NEIL1 as the proof-of-principle DNA glycosylase, we developed a fluorescence-based assay that utilizes incision of site-specifically modified oligodeoxynucleotides to detect enzymatic activity. Among the top hits of these screens were several purine analogs, whose postulated presence in the active site of NEIL1 was consistent with the paradigm of NEIL1 recognition and excision of purine-derived lesions. We also applied gas chromatography/isotope-dilution tandem mass spectrometry for the measurement of the effect of preferred small molecule inhibitors on activities of the DNA glycosylases NEIL1, OGG1 and NTH1. The release of modified bases, which are known substrates of these enzymes, was measured in the presence and absence of the inhibitors. These data revealed that several of the purine analogs were general glycosylase inhibitors with one compound being the most effective inhibitor for all three enzymes. However, there were significance differences in inhibition of enzymatic activities among these three DNA glycosylases. Overall, this work forms the foundation for the future discovery of DNA repair inhibitors as drugs in cancer therapy for the entire family of DNA glycosylases.

Citation Format: Miral Dizdaroglu, Pawel Jaruga. Small molecule inhibitors of DNA glycosylases as potential drugs in cancer therapy. [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 2421. doi:10.1158/1538-7445.AM2014-2421
Abstract 2435: Novel regulators of renal cancer metabolism
Authors: Kho EunYoung , Kirkman Richard , Shim Eun-Hee , Goash Arindam , Sudarshan Sunil
Published: May 2015

Renal cell carcinoma (RCC) is among the 10 most common malignancies in the U.S. Clear cell RCC (ccRCC) is the most common histology. Recent analysis of ccRCC by the Cancer Genome Atlas (TCGA) demonstrates the reduced mRNA expression of several enzymes of the tricarboxylic acid (TCA) cycle. Moreover, reduced TCA enzymes expression is associated with worse prognosis. These data indicate a transcriptional program that promotes a bioenergetic shift. These studies prompted us to examine transcription factors that are known regulators of mitochondrial metabolism including the estrogen-related receptors (ERRs) and peroxisome proliferator-activated receptor γ (PPARγ) coactivators 1 (PGC-1s). Interestingly, mRNA analysis of RCC tissues and cell lines demonstrates the reduced expression of ERRγ and PGC-1α. Notably, prior analysis demonstrates putative ERR binding sites in the promoters of FH and MDH2, two genes encoding TCA enzymes with reduced expression in ccRCC. Promoter analysis of these genes with a luciferase reporter assay demonstrates that ERRγ and PGC-1α cooperate to regulate the expression of TCA enzymes indicating that reduced expression of these factors promotes reduced TCA enzyme expression in RCC.

Citation Format: EunYoung Kho, Richard Kirkman, Eun-Hee Shim, Arindam Goash, Sunil Sudarshan. Novel regulators of renal cancer metabolism. [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 2435. doi:10.1158/1538-7445.AM2014-2435
Abstract 2436: Regulation of breast cancer cell metabolism by the AMPK/ERR/PGC pathway
Authors: Audet-Walsh Étienne , Papadopoli David , St-Pierre Julie , Giguère Vincent
Published: May 2015

Reprogramming of energy metabolism is now considered a hallmark of cancer. Deciphering molecular mechanisms underlying this process is therefore necessary to understand malignant transformation and cancer progression, but also to develop novel therapeutic tools. In the present study, our aim was to discover potential molecular links between two major pathways involved in energy metabolism in breast cancer cells: the energy sensor AMPK (AMP-activated protein kinase) in relationship with the estrogen-related receptors (ERRα and ERRγ) and their coactivators PGC-1α and PGC-1β (peroxisome-proliferator activated receptor γ coactivator-1). Using microarray analysis and qRT-PCR validation, we showed that activation of AMPK significantly altered the expression of the ERRs and the PGCs as well as their metabolic target genes. Moreover, pathway analysis revealed that a significant subset of genes regulated by AMPK pharmacological activation for 1 and 4 days were associated with the ERR/PGC transcriptional programs. Using metabolomics tools, we demonstrated that the energetic remodeling following AMPK pharmacological activation depends in part on ERR/PGC in breast cancer cells. Indeed, the induction in glucose and glutamine uptake and usage by activated AMPK was decreased by half following impairment of the ERR/PGC axis. Moreover, total, uncoupled and coupled respirations were also significantly decreased by ≈50% following ERR/PGC siRNA-mediated knock down. In order to understand the role of the ERRs in the transcriptional regulation of downstream targets of AMPK, we performed chromatin immunoprecipitation followed by massively parallel DNA sequencing (ChIP-seq). Our results indicate that AMPK activation induces ERRs binding at the genomic scale, with the presence of 12,043 and 8,559 novel binding sites for ERRα and ERRγ, respectively. Basal, ERRs' binding to energy metabolism genes was already strong and did not change significantly after treatment. However, AMPK pharmacological activation induced ERRs binding to novel pathways such as numerous genes of the folate metabolism pathway. The biological consequence of the modulation of these pathways by the AMPK/ERR/PGC axis is presently under investigation. In conclusion, using transcriptomics, metabolomics and functional genomics, we revealed the interconnection of the AMPK with the ERR/PGC axis and the importance of this pathway in breast cancer cell metabolic states.

Citation Format: Étienne Audet-Walsh, David Papadopoli, Julie St-Pierre, Vincent Giguère. Regulation of breast cancer cell metabolism by the AMPK/ERR/PGC pathway. [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 2436. doi:10.1158/1538-7445.AM2014-2436
Abstract 2440: RNF5 mediates ER stress-induced degradation of SLC1A5 in breast cancer
Authors: Jeon Young Joo , Khelifa Sihem , Feng Yongmei , Lau Eric , Cardiff Robert , Kim Hyungsoo , Rimm David L. , Kluger Yuval , Ronai Ze'ev
Published: May 2015

Tumor cells have an altered metabolism that distinguishes it from their normal counterpart. Intriguingly, tumor cells show increased use of glutamine to fuel catabolic processes. Therefore, altered metabolic tumors have been considered as a good target for anticancer therapy. Ring finger protein 5 (RNF5) is an ER membrane-associated ubiquitin E3 ligase. RNF5 expression has been associated with breast cancer progression. We identified solute carrier family 1 member 5 (SLC1A5) as a possible RNF5 regulated protein using a MS analysis of breast cancer cells. SLC1A5 is a high affinity L-glutamine transporter, which is highly expressed in tumors such as breast, liver and lung. RNF5 promotes ubiquitination-mediated degradation of SLC1A5 in response to chemotherapeutic drugs known to induce ER stress. RNF5-mediated SLC1A5 instability attenuated mTOR signaling, leading to inhibition of tumor cell proliferation and promotion of apoptosis/autophagy. The loss of RNF5 in PyMT-MMTV crossed with RNF5 mutant animals developed less differentiated mammary tumors, which exhibit increased levels of SLC1A5. TMA analysis of human breast cancer tumors identified a fraction that exhibited an inverse correlation between RNF5 and SLC1A5 expression. Notably, reduced expression of SLC1A5 exhibited a strong association with improved prognosis of breast cancer. Taken together, our observations identify mechanisms underlying the control of SLC1A5 stability, and the implications of such regulation for mTOR signaling and breast cancer survival in response to therapy.

Citation Format: Young Joo Jeon, Sihem Khelifa, Yongmei Feng, Eric Lau, Robert Cardiff, Hyungsoo Kim, David L. Rimm, Yuval Kluger, Ze'ev Ronai. RNF5 mediates ER stress-induced degradation of SLC1A5 in breast 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 2440. doi:10.1158/1538-7445.AM2014-2440
Abstract 2455: BRCA1 and BARD1 expression and localization in breast cell lines
Authors: Herrera Cristobal , Pérez Elisa , Blanco Elías , Gajardo Patricia , Carvallo Pilar
Published: May 2015

One of the principal functions of BRCA1 is to keep the integrity of DNA by homologous recombination which repairs DNA double strand breaks, thus its localization is mainly nuclear in a normal cell. BRCA1 nuclear function is enhanced by its interaction with BARD1, which promotes the import and nuclear retention of BRCA1 by masking its nuclear export signal. Therefore, the interaction of these two proteins is relevant in BRCA1 nuclear function. On the other hand, in tumor cell lines, splicing variants of BARD1 lacking the BRCA1 interaction domain have been described. The expression of these splicing variants of BARD1 could contribute to the loss of nuclear localization of BRCA1. Recently, our group found that a high percentage of triple negative breast cancer tumors showed cytoplasmic or no expression of BRCA1 (unpublished data). Our interest is to study the molecular mechanisms that explain the cytoplasmic localization of BRCA1 in breast cancer cells. We studied mRNA expression of BRCA1, BARD1 and BARD1β through RT-PCR and expression of both proteins by western blot, immunocytochemistry and immunofluorescence in two breast cancer cell lines (HCC1937: ER-, mutated in BRCA1, and T47D: ER+) and one non-tumor breast cell line (MCF10A). BRCA1 and BARD1 expression was coherent between immunocytochemistry and immunofluorescence assay. The HCC1937 cell line presented a weak BRCA1 protein expression mainly in the cytoplasm. The other cell lines had a high nuclear and weak cytoplasmic expression. BARD1 protein is expressed in the nucleus and cytoplasm in all cell lines. BARD1 full mRNA had an equal expression in the three cell lines, and its isoform BARD1β, which do not interact with BRCA1, was expressed at higher levels in tumor cell lines than in the non-tumor cell lines. However, T47D cell line showed nuclear localization of BRCA1 despite high levels of BARD1β mRNA. This suggests that BARD1β either does not express at protein level, or that full-length BARD1 levels are enough to keep BRCA1 in the nucleus. The study of molecular causes of BRCA1 localization in cell lines will allow us to better understand subcellular localization of BRCA1 in tumors and its interaction with BARD1. FONDECYT 1120200.

Citation Format: Cristobal Herrera, Elisa Pérez, Elías Blanco, Patricia Gajardo, Pilar Carvallo. BRCA1 and BARD1 expression and localization in breast cell lines. [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 2455. doi:10.1158/1538-7445.AM2014-2455
Abstract 2466: The tumor suppressor Merlin decreases TGFβ/Smad signaling in metastatic breast cancer cells
Authors: Jackson William P. , Kreitzburg Kelly M. , Das Shamik , Bailey Sarah K. , Metge Brandon J. , Samant Rajeev S. , Shevde-Samant Lalita R.
Published: May 2015

Background: Merlin is a tumor suppressor protein encoded by the NF2 gene. Mutations in NF2 have been implicated in neurofibromas, schwannomas, and mesothelioma. While the mutation rate of NF2 is low in breast cancer, our lab has previously demonstrated a loss of the Merlin protein in advanced stage breast cancers. We have shown that the matricellular metastasis-promoting protein osteopontin activates Akt which then marks Merlin for targeted proteasomal degradation. In the context of other cancers, Merlin has been shown to play a role in sensitization to contact inhibition, cell proliferation, inhibiting invasion, and the negative regulation of focal adhesion kinase. Merlin is also an upstream regulator of the Hippo pathway. However, the implications of the loss of Merlin in the context of breast cancer signaling pathways that promote tumor progression and metastasis have not been fully explored. Here we demonstrate a Merlin-dependent decrease in TGFβ signaling in metastatic breast cancer cells.

Methods: Using Ingenuity Pathway Analysis we analyzed a gene expression array comparing the triple-negative metastatic SUM159 breast cancer cells and their counterparts that were stably restored for Merlin expression. TGFβ was identified as a significant upstream regulator. TGFβ signaling was analyzed in these cells via a SBE4-luc luciferase assay. Signaling was further analyzed by Western blots for SMAD and phospho-SMAD proteins.

Results and Conclusions: Re-expression of Merlin in metastatic breast cancer cells with low-levels of endogenous Merlin decreases TGFβ signaling, demonstrated by decreased luciferase assay activity, decreased rSMAD expression, decreased coSMAD expression and increased iSMAD protein levels. Future studies will explore the nature of this Merlin-dependent decrease in TGFβ signaling.

Acknowledgments: R01CA138850 to L. A. Shevde

Citation Format: William P. Jackson, Kelly M. Kreitzburg, Shamik Das, Sarah K. Bailey, Brandon J. Metge, Rajeev S. Samant, Lalita R. Shevde-Samant. The tumor suppressor Merlin decreases TGFβ/Smad signaling in metastatic breast cancer 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 2466. doi:10.1158/1538-7445.AM2014-2466
Abstract 2514: Towards homogenous adcs: A new site-specific antibody conjugation using bacterial transglutaminase (btg-adc)
Authors: Lhospice Florence , Bregeon Delphine , Belmant Christian , Represa Agnes , Boedec Angelique , Morel Yannis , Dennler Patrick , Schibli Roger , Romagne François
Published: May 2015

Purpose : Bacterial Transglutaminase (BTG) allows coupling on endogenous Q295 from an aglycosylated antibody. Here, the work relates to ADCs synthesis through BTG approach on mAbs with single point mutations and the study of stability, pharmacokinetics, as well as in vitro and in vivo efficacy of resulting BTG-ADCs. AdcetriS®, brentuximab vedotin, has been used as comparator, hence for consistency BTG-ADCs have been conjugated with the different linkage strategies (one step and two step) containing distally the same protease sensitive moiety compared to ADCETRIS®, i.e. valine-citrulline-PAB-MMAE.

Methods : . For synthesis, a two-step process has been designed consisting of coupling first a linker and coming back with a toxin, and affording versatility and efficiency. The DAR stability was monitored over one week in human and cynomolgus plasma by affinity capture/LC/MS. The pharmacokinetic profile was studied in rat and monitored by affinity capture/LC/MS for conjugated antibody and ELISA for total antibody. In vitro efficacy of ADCs conjugated using BTG was compared side-by-side with ADCETRIS® on Karpas 299 cells. In vivo efficacy was studied in xenogenic nude mice engrafted with Karpas 299 in SC with BTG ADCs or ADCETRIS at doses of 0.6mg/kg.

Results: BTG two-step process leads to ADCs with DAR of exactly 2.0 or 4.0 for antibodies with N297S or N297Q single point mutation respectively. The BTG ADCs were stable in human and cynomolgus plasma, with no degradation observed over 15 days in rats and furthermore with favorable total antibody clearance. In vitro efficacy for BTG ADCs DAR=4.0 was comparable to ADCETRIS (mean DAR=4) with respect to EC50 and plateau. In vivo efficacy for BTG ADC was demonstrated to be at least equivalent to ADCETRIS®.

Conclusions: Pre-clinical proof of concept is demonstrated for BTG coupling. When considering versatility, speed and efficiency of the process, BTG-ADC coupling should provide a promising technology for the next generation of homogeneous ADCs.

Citation Format: Florence Lhospice, Delphine Bregeon, Christian Belmant, Agnes Represa, Angelique Boedec, Yannis Morel, Patrick Dennler, Roger Schibli, François Romagne. Towards homogenous adcs: A new site-specific antibody conjugation using bacterial transglutaminase (btg-adc). [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 2514. doi:10.1158/1538-7445.AM2014-2514
Abstract 2947: Regulation of PD-L1 expression in human melanoma by NF-kB
Authors: Gowrishankar Kavitha , Gunatilake Dilini , Gallagher Stuart , Tiffen Jessamy , Hersey Peter
Published: May 2015

Background : Antibodies targeting the immune check point inhibitor programmed death receptor 1 (PD-1) and its ligand (PD-L1) have been associated with relatively high response rates that appear durable in most patients. Past studies have shown the percentage of melanoma expressing the ligand for PD1, PD-L1 varies from 38 to 53% depending on the cutoff used to determine positivity and the antibodies used. In the majority of melanoma, expression of PD-L1 was associated with tumor infiltrating lymphocytes (TIL) and was believed to be induced by IFN gamma from T cells. Relatively few melanomas had (constitutive) PD-L1 expression in the absence of TILs.

Study and Results: In the present study we examined the signal pathways involved in inducible and constitutive expression of PD-L1 expression on cultured melanoma cells, including those derived from patients pre and post vemurafenib treatment. Our results suggest that nuclear factor kappa B (NF-kB) has a role in IFN-gamma inducible expression as shown by inhibition of PD-L1 expression with the inhibitor, BMS 345541 which blocks phosphorylation of the inhibitory IkB kinase, resulting in cytoplasmic retention of NF-kB p65 and p50 subunits. Luciferase based promoter reporter assays confirmed an association between NF-kB activity and PD-L1 expression levels. Targeting NF-kB was more effective in reducing inducible PD-L1 levels than targeting either the MAPK pathway or the P13Kinase/AKT pathway. Transfections with the super repressor, Inhibitor of kappa B alpha, mutated at S32 and S36, to correlate with PD-L1 levels are being carried out. In addition, si RNA mediated knockdown of the p50 and p65 subunits to confirm the involvement of NF-kB in PD-L1 expression will be studied. We are also currently dissecting the signaling pathways involved in constitutive expression of PD-L1.

NF-kB is an important protein complex involved in adaptive and innate immune response and regulates cytokine and chemokine production in melanoma. It also contributes to induction of proteins involved in cell proliferation and survival, playing a role in resistance to targeted therapy. The present results highlight another important function of this crucial transcription factor which our recent studies show may be targetable by inhibitors of the bromo domain and terminal repeat (BET) reader proteins.

Citation Format: Kavitha Gowrishankar, Dilini Gunatilake, Stuart Gallagher, Jessamy Tiffen, Peter Hersey. Regulation of PD-L1 expression in human melanoma by NF-kB. [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 2947. doi:10.1158/1538-7445.AM2014-2947
Abstract 295: Urine and serum biomarkers of pancreatic cancer
Authors: Nolen Brian M. , Grizzle William , Brand Randall , Lokshin Anna E.
Published: May 2015

The clinical management of pancreatic cancer is severely hampered by the absence of effective screening tools. Sixty seven candidate biomarkers were evaluated in prediagnostic sera obtained from cases of pancreatic cancer enrolled in the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial (PLCO). The multimarker panel of CA 19-9, OPN, and OPG, previously shown to be useful in retrospective samples, demonstrated markedly diminished performance in the PLCO cohort. CA 19-9, CEA, NSE, bHCG, and CEACAM1were significantly elevated in sera obtained from cases greater than 1 year prior to diagnosis. Levels of CA 19-9, CA 125, CEA, PRL, and IL-8 were negatively correlated with time to diagnosis. When the entire PLCO set was used for training at a specificity (SP) of 95%, a panel consisting of CA 19-9, CEA, and Cyfra 21-1 provided sensitivity (SN) levels of 33.3% in samples collected >1 year prior to diagnosis compared to SN levels of 23.8% for CA 19-9 alone in the same sample sets. Further study performed using pooled PLCO samples identified 25 candidate biomarkers demonstrating significantly altered expression in pre-diagnostic serum samples. Robust performance of 3 of these biomarkers, GM-CSF, fibronectin, HCG, and SHBG was validated in individual PLCO samples. Next, performance of most promising serum biomarkers was assessed in case/control urine samples and in urine samples from a prospective Shanghai clinical trial. In each setup, better performance of urine biomarkers in comparison with serum counterparts was determined. A biomarker panel comprised of urine CA 19-9, leptin, OPN, and IL-8 had a SN=39% at a SP of 95% in Shanghai samples. These results warrant further studies to develop early detection tests for PDAC with high levels of sensitivity.

Note: This abstract was not presented at the meeting.

Citation Format: Brian M. Nolen, William Grizzle, Randall Brand, Anna E. Lokshin. Urine and serum biomarkers of pancreatic 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 295. doi:10.1158/1538-7445.AM2014-295
Abstract 2959: The tumor suppressor CHD5 is an epigenetic regulator of neuronal cell fate
Authors: Mills Alea A. , Hwang Dong-Woo
Published: May 2015

Genomic lesions affecting the 1p36 chromosomal region are prevalent in variety of human malignancies, yet the tumor suppressor in this region had not been identified. We used chromosome engineering to create a series of mouse models with lesions spanning the 1p36-analagous region. These gain and loss-of-function models allowed us to pinpoint a potent tumor suppressive interval that when deleted predisposes to cancer and when duplicated causes excessive tumor suppression. We identified Chromodomain Helicase DNA-binding domain protein 5 (CHD5) as the causative gene in the region, defined CHD5 as an inducer of the tumor suppressive network encoded at the Cdkn2 locus, and demonstrated that CHD5 is frequently deleted in human glioma. It is now appreciated that CHD5 is mutated in cancers of the breast, ovary, colon, and prostate, as well as in melanoma, glioma, and neuroblastoma, and furthermore, that robust CHD5 expression is a favorable indicator of patient survival following anti-cancer therapy.

Although ample evidence from human studies indicates that CHD5 plays a pivotal role in cancer, the mechanism responsible for tumor suppression is not well understood. CHD5 belongs to the CHD family of ATP-dependent chromatin remodeling proteins, yet CHD5's role in modulating chromatin and the consequence of CHD5 deficiency in vivo was unknown. We discovered that CHD5's tumor suppressive function is dependent on its dual plant homeodomains (PHDs)_motifs that bind specifically marked histones. The PHDs of CHD5 preferentially bind unmodified histone 3 (H3), an interaction essential for CHD5's ability to inhibit proliferation, to modulate transcription, and to suppress tumorigenesis in vivo. We recently defined CHD5 as a regulator of chromatin-mediated dynamics essential for neuronal differentiation. Whereas neural stem cells (NSCs) normally have the capacity to differentiate into neurons, NSCs from Chd5-deficient mice retain stem-like characteristics and differentiate into astrocytes at the expense of neurons. We discovered that H3K27me3_a repressive chromatin mark implicated in cancer_is depleted by CHD5 deficiency. We show that CHD5 rescues the de-differentiated phenotype of CHD5 deficient NSCs, while re-establishing global levels of H3K27me3.

Our findings define CHD5 as a dosage-sensitive tumor suppressor, identify CHD5 as a member of a newly appreciated class of H3-binding PHD-containing proteins, and reveal a critical role for CHD5 in dictating stem cell fate. This provides new insight into the epigenetic mechanisms responsible for CHD5's tumor suppressive role that when perturbed, set the stage for cancer.

Citation Format: Alea A. Mills, Dong-Woo Hwang. The tumor suppressor CHD5 is an epigenetic regulator of neuronal cell fate. [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 2959. doi:10.1158/1538-7445.AM2014-2959
Abstract 2964: The codon 47 Pro47Ser polymorphism influences p53 phosphorylation and apoptotic function
Authors: Jennis Matthew , Hollstein Monica , Murphy Maureen E.
Published: May 2015

The p53 tumor suppressor protein is a central signaling protein in response to oncogenic, genotoxic and metabolic stress. Single nucleotide polymorphisms (SNPs) in p53 can have a marked impact on p53 tumor suppressor function, and have been associated with increased cancer risk. Amino acid 47 of p53 is a conserved residue, located within the second transactivation domain of this protein. In wild type p53, this residue is proline (P47, or WT). Importantly, this proline is critical for the ability of proline-directed kinases to phosphorylate p53 on the adjacent residue, serine 46. Phosphorylation of serine 46 of p53 greatly enhances the ability of p53 to transactivate certain target genes, and to induce apoptosis. In some Africans and African Americans, amino acid 47 of p53 encodes serine (S47) instead of proline. We have shown that the S47 polymorphism eliminates phosphorylation on serine 46, and reduces p53's ability to induce apoptosis by at least 3-fold. We have created mice that contain the humanized p53 gene (Hupki) for either the S47 or WT alleles of p53. In new data, we show that tissues from these mice have significantly decreased apoptosis after gamma radiation. We also show that apoptosis induced by cisplatin is markedly defective in S47 mice and cell lines. Further, MEFs from S47 mice have a 15-fold increase in IC50 for cisplatin, indicating that this polymorphism will impact the efficacy of cisplatin therapy in Africans and African Americans. Finally, we have identified two critical p53 target genes, GLS2 (metabolism) and NOXA (apoptosis), which show significantly impaired transactivation by S47. The long-term objective of this research is to further characterize the function of the S47 variant, and to identify the contribution of this variant to cancer risk and progression in Africans and African Americans.

Citation Format: Matthew Jennis, Monica Hollstein, Maureen E. Murphy. The codon 47 Pro47Ser polymorphism influences p53 phosphorylation and apoptotic function. [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 2964. doi:10.1158/1538-7445.AM2014-2964
Abstract 2975: The pluripotency factor LIN28 promotes colorectal tumorigenesis and tumor progression
Authors: Schwitalla Sarah , Tu Ho-Chou , Viswanathan Srinivas , Zhu Hao , Qian Zhirong , Shah Samar , Ogino Shuji , Daley George Q.
Published: May 2015

LIN28A and LIN28B are RNA-binding protein paralogs expressed in embryonic stem cells and in early developing tissues. Through blocking the terminal processing of let-7 miRNAs and regulating the translation of a number of mRNA binding targets, LIN28 controls an array of targets that mediate proliferation, pluripotency, differentiation and metabolism. While highly expressed in the early embryos, the levels of LIN28A/B are downregulated as differentiation and development proceed. However, aberrant overexpression of LIN28 is often observed in human malignancies especially those with poor prognosis. We have demonstrated that LIN28 promotes cellular transformation in vitro, yet it is unknown how LIN28 regulates tumorigenesis in vivo. Here, we investigate the role of LIN28 in colorectal cancer (CRC) in humans and in mouse models. We first investigated the status of LIN28A/B in 1000 CRC patients and observed 35-60% of the cancer samples express LIN28A and/or LIN28B. Furthermore, we generated intestinal-specific LIN28 overexpressing mice, and observed intestinal adenoma as well as invasive adenocarcinoma that highly resemble the histology of human CRCs. Moreover, in Apcmin/+ animals, additional LIN28 expression significantly increased tumor incidence and accelerated disease onset, as their tumors are poorly differentiated and more invasive compared with Apcmin/+ tumors. Together, our study shows that LIN28 drives intestinal tumorigenesis and further promotes tumor progression in vivo. CRC is one of the major contributors to cancer-related mortality; however, current mouse models seldom progress to later stages of the disease, which greatly impedes efforts to developing more effective therapies. Based on the knowledge gained from this research, we hope to provide novel insights into designing therapeutics targeting late-stage CRC.

Citation Format: Sarah Schwitalla, Ho-Chou Tu, Srinivas Viswanathan, Hao Zhu, Zhirong Qian, Samar Shah, Shuji Ogino, George Q. Daley. The pluripotency factor LIN28 promotes colorectal tumorigenesis and tumor progression. [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 2975. doi:10.1158/1538-7445.AM2014-2975
Abstract 2992: Zoledronic acid inhibits angiogenesis through Rac1 inactivation in ovarian cancer
Authors: Gonzalez-Villasana Viany , Rodriguez-Aguayo Cristian , Fuentes-Mattei Enrique , Pradeep Sunila , Ivan Cristina , Sood Anil K. , Lopez-Berestein Gabriel
Published: May 2015

Background Angiogenesis, the formation of new blood vessels from pre-existing vasculature, is a complex and crucial process for tumor growth and development of metastasis. Rac1, a member of the Rho GTPase family, plays a central role in angiogenesis through regulation of endothelial cell migration, tube formation, adhesion, invasion, and proliferation. In this study we hypothesized that zoledronic acid (ZA) inhibits ovarian cancer angiogenesis through the inactivation of Rac1. Methods We treated HeyA8 MDR and Ovcar-5 cells with ZA to assess its effects on cell invasion, proliferation, expression of matrix metalloproteinases (MMPs; Milliplex assay) and angiogenic factors. We also determined Rac1 activation by pull-down assay. HeyA8 MDR and Ovcar-5 orthotopic murine models of ovarian cancer were assessed for expression of CD31 and Ki-67, Rac1 activation, and apoptosis by immunohistochemistry after treatment with ZA, nanoparticle, albumin-bound paclitaxel (nab-paclitaxel), or a combination. Results In vitro, we observed inhibition of HeyA8 MDR and Ovcar-5 cell invasion and proliferation by ZA. Moreover, ZA decreased the secretion of MMP-7 and vascular endothelial growth factor C (VEGF-C) in HeyA8 MDR cells and MMP-9, MMP-10, VEGF-C, granulocyte-colony stimulating factor, endothelin-1, follistatin, interleukin-8, placenta growth factor, fibroblast growth factor-1, and hepatocyte growth factor in Ovcar-5 cells. Furthermore, ZA inactivated Rac1 in both cells lines. In vivo, ZA decreased the number of nodules and tumor weight, inhibited angiogenesis and cell proliferation, and increased apoptosis in both HeyA8 MDR and Ovcar-5 animal models. These in vivo antitumor effects were enhanced in both models when ZA was combined with nab-paclitaxel. Conclusion ZA inhibits ovarian cancer angiogenesis through the inactivation of Rac1, suggesting that ZA combined with paclitaxel could be a novel therapeutic approach to ovarian cancer angiogenesis.

Citation Format: Viany Gonzalez-Villasana, Cristian Rodriguez-Aguayo, Enrique Fuentes-Mattei, Sunila Pradeep, Cristina Ivan, Anil K. Sood, Gabriel Lopez-Berestein. Zoledronic acid inhibits angiogenesis through Rac1 inactivation in ovarian 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 2992. doi:10.1158/1538-7445.AM2014-2992