bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract KP01: Identification of genes and pathways that prevent genome instability
Authors: Kolodner Richard
Published: August 2015

The genome of many cancers is characterized by ongoing genome instability; however, the causes of genome instability in cancer are not well understood. To understand the control genome instability, we developed three types of genetic assays for measuring the rate of accumulating gross chromosomal rearrangements (GCRs) in the yeast Saccharomyces cerevisiae - one that detects GCRs containing breakpoints in single copy sequences, a second that detects GCRs mediated by segmental duplications, and a third that detects GCRs mediated by Ty elements which are similar to mammalian LINE elements. Initial studies characterizing these three types of assays found that there are specific pathways that suppress duplication-mediated GCRs and identified sequences that are hotspots for the formation of GCRs. We have subsequently used these three assays to perform a genetic screen of the non-essential genes in S. cerevisiae to identify the genetic network that acts to prevent genome instability. This screen involved three steps: a novel bioinformatics screen to identify and prioritize candidate genome instability suppressing genes; a biological genetic screen to identify which of the predicted genes actually suppress genome instability; and a biological genetic screen to identify interacting genes. As a result of these screens, we have identified 184 genes in which defects cause increased GCRs and an additional 459 genes in which mutations only cause increased GCRs when combined with a second mutation thus defining multiple pathways that prevent GCRs. Control experiments indicated that this approach led to the identification of greater than 95% of the non-essential genes that act to suppress genome instability. In a second screen, we identified 44 essential genes in which defects caused increased GCRs. We are now beginning to use the S. cerevisiae genome instability suppressing genes to predict the mammalian genome instability genetic suppressing network.

Citation Format: Richard Kolodner, PhD. Identification of genes and pathways that prevent genome instability [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr KP01.
Abstract POSTER-BIOL-1303: ARID3B alters tumor cell adhesion by binding to the promoter regions in fzd5 and other wnt pathway genes
Authors: Bobbs Alexander , Hallas William Morgan , Gellerman Katrina , Joseph Stancy , Dahl Karen Cowden
Published: August 2015

ARID3B is a DNA-binding protein from the AT-rich interactive domain family that is overexpressed in ovarian cancer and promotes tumor progression in a xenograft model of ovarian cancer in nude mice. Prior to this study, direct ARID3B target genes had not been identified, and relatively little was known about ARID3B's function or consensus binding site. In this study, we identified and confirmed the first gene targets of ARID3B by chromatin immunoprecipitation, microarray, and quantitative real-time PCR. Based on the binding sites that were determined in this study, we defined a novel ARID3B binding site that was tested in an electrophoretic mobility shift assay. Analysis of ARID3B binding regions revealed that ARID3B regulates genes in key cellular pathways that are involved in cancer progression and drug resistance, including EGFR, NOTCH, TNF, centromere proteins, and the Wnt signaling pathway. We have focused on Wnt signaling, and showed that ARID3B binds to the promoter regions of Wnt5A and its receptor FZD5, along with downstream components of the pathway: APC and MYC. FZD5 is commonly over-expressed in ovarian cancer cell lines, and is further upregulated by excess ARID3B. Upregulation of either ARID3B or FZD5 increased adhesion to extra-cellular matrix components Collagen IV, Fibronectin, Laminin, Tenascin, and Vitronectin. These results establish that Wnt signaling is targeted by ARID3B, and provide key insights into how ARID3B affects tumor progression.

Citation Format: Alexander Bobbs, William Morgan Hallas, Katrina Gellerman, Stancy Joseph Karen Cowden Dahl. ARID3B alters tumor cell adhesion by binding to the promoter regions in fzd5 and other wnt pathway genes [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-BIOL-1303.
Abstract POSTER-BIOL-1306: DNA methylation changes during epithelial-to-mesenchymal transition in ovarian cancer cells
Authors: Cardenas Horacio , Vieth Edyta , Lee Jiyoon , Liu Yunlong , Nephew Kenneth P. , Matei Daniela
Published: August 2015

Epithelial-to-mesenchymal transition (EMT) is an important step in the process of metastasis and has been linked to cancer cell stemness. We hypothesize that aside from defined genetic changes associated with EMT, epigenetic mechanisms are involved and have not been fully elucidated. We determined DNA methylation in SKOV3 ovarian cancer (OC) cells during EMT induced by TGF-ß using the Infinium HumanMethylation450 array. Methylation levels of CpG sites were expressed as ß-values ranging from 0 (totally unmethylated) to 1 (totally methylated). Methylation of 468 and 390 sites changed (P<.01, ANOVA and FDR<0.05) after 48h and 120h of TGF-ß stimulation. The majority of changes (81%) reflected TGF-ß-induced increase in methylation. Methylation of 160 sites changed similarly at 48 and 120 h indicating that 34% of initial changes were sustained. The number of methylation changes decreased significantly after TGF-ß removal from the cell cultures, demonstrating that removal of TGF-ß stimulation reversed the methylation changes at most sites and restituted the baseline methylome. These results were consistent with those of the principal component and hierarchical clustering analyses. TGF-ß increased overall (average of all CpG sites) DNA methylation, total DNA methyltransferase activity, and amounts of DNMT1 protein and mRNA. The average methylation of CpG islands located in or near promoters increased in 193 genes at 24h and 494 at 120h, while no changes were observed after TGF-ß removal. Pathway analysis identified methylation changes in genes belonging to functional networks relevant to EMT and cancer progression, including: cellular movement, cell cycle, organ morphology, cellular development, and cell death and survival. Treatment of OC cells with the DNA methylation inhibitor SGI-110 prevented TGF-ß-induced EMT in association with an inhibition of the TGF-ß-induced downregulation of E-Cadherin. Results demonstrate that DNA methylation is a dynamic process involved in regulation of genes implicated in EMT and metastasis.

Citation Format: Horacio Cardenas, Edyta Vieth, Jiyoon Lee, Yunlong Liu, Kenneth P. Nephew, Daniela Matei. DNA methylation changes during epithelial-to-mesenchymal transition in ovarian cancer cells [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-BIOL-1306.
Abstract POSTER-BIOL-1331: Nucleotide excision repair mutations confer sensitivity to platinum but not PARP-inhibitors in epithelial ovarian cancer
Authors: Ceccaldi Raphael , O'Connor Kevin , Mouw Kent W. , Li Adam Y. , Matulonis Ursula A. , D'Andrea Alan D. , Konstantinopoulos Panagiotis A.
Published: August 2015

The marked platinum sensitivity of epithelial ovarian cancer (EOC) is thought to be related to underlying defects in the homologous recombination (HR) DNA repair pathway, which are present in approximately 50% of tumors with high-grade serous histology. However, repair of platinum-induced DNA damage does not always involve the HR pathway. More than 90% of platinum-DNA lesions are intrastrand cross-links which are repaired by the nucleotide excision repair (NER) pathway. We curated the TCGA ovarian cancer dataset and identified 22 tumors (7%) with nonsynonymous or splice site NER gene mutations among the 316 sequenced high grade serous ovarian tumors. All NER mutations were mutually exclusive and 8 (36%) were accompanied by heterozygous loss of the respective NER gene. In 12 (55%) of the 22 cases, there were no concomitant HR alterations identified. In vitro functional evaluation of two NER mutations (ERCC6-Q524* and ERCC4-A583T), identified in patients with advanced stage tumors and extreme response to first-line platinum therapy, showed that both mutations conferred platinum, but not PARPi, or doxorubicin sensitivity, and did not affect HR activity. Interestingly, ERCC6-Q524* was not associated with heterozygous loss but sensitized cells to platinum in a dominant negative fashion. Our findings suggest a novel mechanism of platinum sensitivity in EOC that involves NER gene mutations, which may occur independently of HR alterations. Unlike tumors with defective HR, tumors harboring only NER mutations may be sensitive to platinum but not PARP inhibition. Dissociation of platinum and PARPi sensitivity in this setting may have important implications for PARPi trials for both HR-proficient and HR-deficient EOCs.

Citation Format: Raphael Ceccaldi, Kevin O’Connor, Kent W. Mouw, Adam Y. Li, Ursula A. Matulonis, Alan D. D’Andrea, Panagiotis A. Konstantinopoulos. Nucleotide excision repair mutations confer sensitivity to platinum but not PARP-inhibitors in epithelial ovarian cancer [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-BIOL-1331.
Abstract POSTER-BIOL-1337: Omentum promotes suppression against peritoneal tumors
Authors: Meza-Perez Selene , Garcia-Hernandez Maria de la Luz , Silva-Sanchez Aaron , Rangel-Moreno Javier , Mudunuru Uma , Lord Edith M , Randall Troy D.
Published: August 2015

Purpose: The metastasis of ovarian cancer to the omentum is associated with poor clinical outcomes. Although the omentum has immune function due to the activities of milky spots, the role of the omentum in anti-tumor immunity has not been rigorously addressed. We hypothesize that the omentum directly modulates anti-tumor immunity to metastasized tumor cells.

Experimental procedure: We examined metastases and immunological function in mice that expresses SV40 T antigen oncogene under the Mullerian Inhibitory Substance type II Receptor promoter (MISIIR-Tag). In separate experiments we also implanted 3 x 106 EG7.1.15 cells (murine thymoma that express chicken ovalbumin (OVA)) intraperitoneally (i.p.) to induce peritoneal tumors. We used flow cytometry to analyze cells from the omentum and peritoneal exudate from mice with or without tumors.

Results: Omental tumor growth induced by EG7.1.15 cells is associated with an increase in CD4+CD25+FoxP3+ T regulatory cells (Tregs) and CD103+ DCs, while tumor-specific CD8+ T cells are reduced. Although tumor-specific CD8+ T cells generated in the periphery prior to tumor onset prevent omental and peritoneal tumor growth, tumor-specific CD8+ T cells generated after tumors are established in the omentum are unable to mediate tumor clearance. The induction of tolerance requires the omentum, is dependent on FoxP3+ Tregs, is tumor antigen-specific and takes place in as few as 6 days. The metastasis of ovarian tumor cells into the omentum also correlates with an increase in Tregs. Deleting Tregs in both models reduces tumor burden.

Conclusions: Metastasis of tumor cells to the omentum leads to immunological tolerance rather than immunity – even when tumor cells express foreign antigens. Thus, the omentum has an immunological activity that prevents, rather than promotes, immunity to peritoneal tumors.

Citation Format: Selene Meza-Perez, Maria de la Luz Garcia-Hernandez, Aaron Silva-Sanchez, Javier Rangel-Moreno, Uma Mudunuru, Edith M Lord PhD, Troy D. Randall. Omentum promotes suppression against peritoneal tumors [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-BIOL-1337.
Abstract POSTER-BIOL-1341: MUC16 (CA125) knockdown as a potential strategy to decrease tumor growth
Authors: Felder Mildred , Kapur Arvinder , Rakhmilevich Alexander L. , Qu Xiaoyi , Connor Joseph , Patankar Manish S.
Published: August 2015

MUC16 (CA125), a membrane spanning mucin, promotes proliferation and metastasis of ovarian cancer cells. Here, we demonstrate that MUC16 protects cancer cells from cytolysis by NK cells and macrophages. SCID mice implanted with MUC16-positive and MUC16-knockdown human ovarian tumors have a median survival of 75 and 181 days, respectively. Animals bearing MUC16-positive tumors developed ascites fluid and were moribund because of high tumor volume. In contrast, none of the mice with MUC16-knockdown tumors developed ascites. Decreased growth of MUC16-knockdown tumors correlated with increased cytolysis by murine splenocytes from naïve and anti-CD40 antibody treated mice. Murine NK cells showed a 2.5-fold higher preference for MUC16-knockdown cells. Unstimulated and LPS or IFN-γ-activated peritoneal macrophages from SCID mice also preferentially inhibited proliferation of MUC16-knockdown targets. Even in SCID/Beige mice, where cytolysis by resident NK cells is impaired, median survival MUC16-positive tumor-bearing animals was 75 days as compared to 189 days for animals with MUC16-knockdown tumors. All ten SCID/Beige mice with MUC16-knockdown tumors showed significant macrophage infiltration throughout the tumor nests and only two of the ten developed ascites. In contrast, 80% of SCID/Beige mice with MUC16-positive tumors had significant ascites and macrophages were predominantly found in the stroma. Finally, knockdown of MUC16 increased the susceptibility of cancer cells to natural cytotoxic responses of human NK cells, and increased their susceptibility to ADCC and lysis in the presence of immunocytokines. Therefore, strategies that decrease the expression of immunosuppressive MUC16 coupled with NK or macrophage-based immunotherapies should be investigated for the treatment of ovarian cancer.

Citation Format: Mildred Felder, Arvinder Kapur, Alexander L. Rakhmilevich, Xiaoyi Qu, Joseph Connor, Manish S. Patankar. MUC16 (CA125) knockdown as a potential strategy to decrease tumor growth [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-BIOL-1341.
Abstract POSTER-BIOL-1344: Epigenetic regulation of SPARC in ovarian cancer
Authors: Matthews Dylan P. , Taylor Sherine , Said Neveen A.
Published: August 2015

Background: We have previously reported the tumor suppressor effect of Secreted protein acidic and rich in cysteine (SPARC) in ovarian cancer in vitro and in vivo. We reported that SPARC suppresses ovarian cancer cells interactions with the surrounding environmental cues within the peritoneal milieu. We also reported that the expression of SPARC was decreased in established ovarian cancer cell lines and micro-dissected ovarian cancer cells from advanced tumors consistent with hypermethylation of its promoter. However, the mechanisms of epigenetic regulation of SPARC in ovarian cancer (and other morbidities) are unraveled.

Experimental Procedures: In silico analysis, human ovarian cancer tissue microarrays, a panel of established human ovarian cancer cell lines and in vitro ovarian cancer cell models were used. Genetic and pharmacologic manipulation of NFκB subunits and DNA methyl transferases (DNMTs) were used to investigate their involvement in the transcriptional regulation of SPARC expression and function.

Results: In silico mapping of SPARC promoter identified consensus binding sites for subunits (RelA and NFκB1). Pharmacologic inhibition of NFκB as well as knockdown of p65-RelA and p50 subunits restored SPARC expression and function in ovarian cancer cells. Treatment of ovarian cancer cells with demethylating agent 5’-azacytidine and knockdown of DNMTs (DNMT1, DNMT3a and DNMT3b) indicated that NFκB-mediated SPARC repression is due to promoter methylation through DNMT3a. In ovarian cancer tissue microarrays, SPARC expression in cancer cells significantly and progressively decreased in advanced stage. The expression of SPARC in cancer cells inversely correlated with nuclear p65-RelA and DNMT3a expression.

Conclusion: Our findings indicate that activated NFκB integrates inflammatory signals to exert epigenetic pressure suppressing tumor-SPARC through promoter methylation. Pharmacologic inhibitors of NFκB as well as anti-inflammatory agents may provide a significant therapeutic opportunity.

Citation Format: Dylan P. Matthews, Sherine Taylor, Neveen A. Said. Epigenetic regulation of SPARC in ovarian cancer [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-BIOL-1344.
Abstract POSTER-CTRL-1208: A dualistic model for the origin of high-grade serous carcinoma: BRCA mutation status, histology and tubal intraepithelial carcinoma
Authors: Howitt Brooke E , Hanamornroongruang Suchanan , Lin Douglas , Conner James E , Schulte Stephanie , Garber Judy , Crum Christopher P , Meserve Emily E
Published: August 2015

Extra-uterine high-grade serous carcinoma (HGSC) is linked to the fimbria via serous tubal intraepithelial carcinoma or STIC. Women with deleterious BRCA1/2 germ-line mutations (BRCA+) are at high risk for HGSC and ~5% of risk reducing salpingo-oophorectomies harbor a STIC. However, STIC is found in only~40% of the HGSC population and less frequently in endometrioid variants of HGSC (Roh 2009). Consecutively tested women with untreated HGSC, with detailed fallopian tube examination (SEE-FIM protocol), who had germ-line BRCA testing were studied. STIC status was determined by histologic exam, and tumors were classified as predominately SET (> 50% solid, endometrioid-like, or transitional, Soslow 2012) or classic histology. SET features trended towards a higher frequency in BRCA+ vs BRCA- women (50 vs 28%, p = .11). BRCA- subjects with SET morphology were significantly younger than those with classic HGSC (mean 56.2 vs 64.8 years; p=0.04), with a generally better clinical outcome. STIC was significantly more frequent in BRCA- tumors (66 vs 31%, p = 0.017) and more frequent in classic HGSCs in BRCA- (83 vs 22%, p = 0.003) women. Overall, several co-variables – histology, BRCA status, age, coexisting STIC, response to therapy - suggest two categories of HGSC with differences in speed of development, progression, outcome, histology, and possibly, precursor type. We introduce a dualistic HGSC model with a faster evolving tumor type that is not linked to a long-standing STIC. Resolving the nature of this second pathway is germane to both resolving the precursors and expectations from both screening and prevention of HGSC.

Citation Format: Brooke E Howitt MD, Suchanan Hanamornroongruang MD, Douglas Lin MD PhD, James E Conner MD PhD, Stephanie Schulte MD, Judy Garber MD, Christopher P Crum MD, Emily E Meserve MD MPH. A dualistic model for the origin of high-grade serous carcinoma: BRCA mutation status, histology and tubal intraepithelial carcinoma [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-CTRL-1208.
Abstract POSTER-CTRL-1217: Human placental lactogen-functionalized bimodal imaging probe for human ovarian cancer detection
Authors: Zhang Yilin , Sundaram Karthik M. , Li Nan-Sheng , Piccirilli Joseph A. , Lengyel Ernst
Published: August 2015

The early detection of ovarian cancer (OvCa) is a challenge because the postmenopausal ovaries are hidden in the peritoneal cavity and current imaging technologies lack the ability to differentiate between benign and malignant masses. Our goal is to improve the detection and imaging of OvCa utilizing cancer cell-targeted, bimodal fluorescent and paramagnetic micelles that effectively detect malignant tumors. The human prolactin receptor (PrlR) is expressed in 98% of malignant ovarian tumors, but is not expressed in benign ovarian tumors or fallopian tubes. Therefore, we conjugated the maleimide-functional micelles to a thiol group of human placental lactogen (hPL), a ligand of PrlR, to specifically target OvCa cells. The micelles were 20-nm in diameter with a favorable surface charge (-5.8mV) characterized by dynamic light scattering, zeta potential, and transmission electron microscopy (TEM). After conjugation to hPL, hPL-micelles displayed an increased molecular weight and particle size as assessed by SDS-PAGE, MALDI-TOF, and size exclusion chromatography. Microscopic and cytometric analyses demonstrated that hPL-micelles were more efficiently internalized in human primary OvCa cells and OvCa cell lines than unconjugated micelles. The knockdown of PrlR using shRNA in OvCa cell lines or the triple mutation of hPL significantly inhibited uptake of hPL-fluorophore conjugates, suggesting receptor-mediated endocytosis. Furthermore, the Xenogen IVIS small animal imaging system detected a greater accumulation of hPL-micelles than of unconjugated micelles in the human ovarian tumor xenograft, confirming the enhanced imaging of OvCa through targeted delivery. Given the low prevalence of OvCa and the current unsatisfactory imaging modalities, “receptor-dependent imaging” through hPL-decorated bimodal micelles has the potential to improve detection of OvCa through enhanced specificity.

Citation Format: Yilin Zhang, Karthik M. Sundaram, Nan-Sheng Li, Joseph A. Piccirilli, Ernst Lengyel. Human placental lactogen-functionalized bimodal imaging probe for human ovarian cancer detection [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-CTRL-1217.
Abstract POSTER-THER-1413: Targeting constitutively-activated STAT3 in hypoxic ovarian cancer
Authors: McCann Georgia A , ElNaggar Adam , Naidu Shan , Rath Kellie S , Tierney Brent J , Suarez Adrian , Cohn David E , Selvendiran Karuppaiyah
Published: August 2015

Background: Tumor hypoxia, a feature of many solid tumors including ovarian cancer, is associated with resistance to therapies. We previously demonstrated that hypoxic exposure results in increased expression of phosphorylated signal transducer and activator of transcription 3 (pSTAT3). We hypothesized that the activation of STAT3 could lead to chemotherapeutic resistance in ovarian cancer cells in hypoxic conditions.

Methods: The increase of pSTAT3 Tyr705 in the hypoxic regions of human epithelial ovarian cancer (EOC) specimens was determined by HIF-1α and CD-31 staining. In vitro mutagenesis studies of pSTAT3 Tyr705 were used to demonstrate the necessity for cell survival and proliferation under hypoxic conditions. In addition, S1PR1, a regulator of STAT3 transcription via the JAK/STAT pathway, is highly expressed in hypoxic ovarian cancer cells (HOCCs). This association was evaluated with knock down of S1PR1 in HOCCs. HOCCs were treated with our novel STAT3 inhibitor, HO-3867.

Results: pSTAT3 Tyr705 is increased in the hypoxic regions of EOC specimens. In vitro mutagenesis studies proved that pSTAT3 Tyr705 is necessary for cell survival and proliferation under hypoxic conditions. Knock down of S1PR1 in HOCCs reduced pSTAT3 Tyr705 levels and was associated with decreased cell survival. Treatment of HOCCs with the STAT3 inhibitor HO-3867 resulted in a rapid and dramatic decrease in pSTAT3 Tyr705 levels as a result of ubiquitin proteasome degradation. There was decreased expression of the STAT3-target proteins Bcl-xL, cyclin D2, and VEGF in all HO-3867 treated cells.

Conclusions: These findings suggest that activation of STAT3 Tyr705 promotes cell survival and proliferation in HOCCs, and that S1PR1 is involved in the initiation of STAT3 activation. Therefore, targeting hypoxia-mediated STAT3 activation represents a therapeutic option for ovarian cancer and other solid tumors.

Citation Format: Georgia A McCann, MD, Adam ElNaggar, MD, Shan Naidu, MS; Kellie S Rath, MD; Brent J Tierney, MD; Adrian Suarez, MD; David E Cohn, MD; Karuppaiyah Selvendiran, PhD. Targeting constitutively-activated STAT3 in hypoxic ovarian cancer [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-THER-1413.
Abstract POSTER-THER-1425: Novel small molecules overcoming platinum drug resistance in ovarian cancer cells
Authors: Qian Wei , Wang Jingnan , Edwards Robert , Wipf Peter , Houten Bennett Van
Published: August 2015

After stage, platinum drug resistance is the single most important factor in determining prognosis in ovarian cancer. Overcoming platinum drug resistance by pharmacological manipulations represents a major challenge in the current clinical practice for the management of ovarian cancer. We discovered a novel drug combination approach using platinum agents with mdivi-1 (mitochondrial division inhibitor 1) that is able to synergistically induce rapid apoptosis in platinum-resistant ovarian cancer cells, including established cisplatin-resistant ovarian cancer cell lines as well as primary cancer cells derived from ovarian cancer patients. We also identified that the synergism between cisplatin and mdivi-1 is not dependent on Drp1 (a mitochondrial fission protein and the reported target of mdivi-1), as mdivi-1 enhances cisplatin toxicity in Drp1 knockout mouse embryonic fibroblast (MEF) cells. In order to improve the compound properties of mdivi-1, we performed a structure activity relationship (SAR) study of mdivi-1 and assembled and screened a small library of structurally related compounds. We were able to identify more potent novel small molecules that synergize with platinum agents. We found that short-term exposure of cells to the combination of cisplatin and those novel compounds is sufficient to reduce cell viability, while cisplatin alone treatment only slightly inhibited cell proliferation in cisplatin-resistant ovarian cancer cells. We further identified that mdivi-1 and its structurally related novel small molecules, when combined with cisplatin, induce a synergistic increase of replication stress and DNA damage, causing upregulation of a pro-apoptotic Bcl-2 family protein Noxa. These small molecules also repress mitochondrial respiration and the combination with cisplatin triggers substantial mitochondrial dysfunction including mitochondrial uncoupling. DNA replication stress and mitochondrial dysfunction converge on the induction of mitochondrial outer membrane permeabilization (MOMP) proceeding robust mitochondrial apoptotic signaling, bypassing a Bax/Bak-dependent mechanism. Since apoptosis induced by cisplatin alone relies on Bax/Bak-dependent MOMP, and inefficient activation of Bax/Bak-dependent MOMP is often associated with cisplatin resistance, the combination of cisplatin and mdivi-1 family compounds represents a novel strategy in overcoming platinum resistance. We thus present a novel platinum-based combination approach in the treatment of platinum resistant ovarian cancer.

Citation Format: Wei Qian, Jingnan Wang, Robert Edwards, Peter Wipf, Bennett Van Houten. Novel small molecules overcoming platinum drug resistance in ovarian cancer cells [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-THER-1425.
Abstract POSTER-THER-1429: Selection of DNA aptamers for an ovarian cancer cell line using high-throughput sequencing
Authors: Whelan Rebecca J. , Kapur Arvinder , Felder Mildred , Shallcross Jamie , Nie Jeff , Patankar Manish S.
Published: August 2015

Humanized antibodies have been extensively investigated as therapeutic as well as diagnostic agents. While the antigen specificity offered by antibodies makes them very attractive for such theranostic applications, their large-scale synthesis can be challenging and expensive. We are therefore investigating alternate strategies to develop agents that can be used for in vivo monitoring as well as for treatment of epithelial ovarian tumors. One approach is to develop Single Stranded DNA aptamers that selectively bind to ovarian cancer cells. The ssDNA aptamers can be synthesized using template-driven or de novo chemical synthetic approaches to manufacture agents at a large scale and low cost. The challenge however, is to develop aptamers that are specific to ovarian cancer cells. In the current study, we report a streamlined approach that incorporates the cell-based Systematic Evolution of Ligands by Exponential Enrichment (cell-SELEX) with DNASeq technology to select aptamers that recognize ovarian cancer cells. An ssDNA aptamer library composed of ~1015 sequences was subjected to ten iterative rounds of selection against the ovarian cancer cell line OVCAR-3. Aptamers from each round were amplified by asymmetric PCR and subjected to high-throughput sequencing. Eight ssDNA aptamers enriched through the selection process were identified by DNASeq and subsequent bioinformatics analysis and their selectivity and affinity for OVCAR-3 cells was determined by flow cytometry. Two of these aptamers (Apt-1 and Apt-8) showed significant binding to OVCAR-3 cells with Kd of 24 and 28 nM, respectively. Secondary structure analysis using mfold indicated that Apt-1 and Apt-8 had defined secondary structures resulting from ordered base pairing of the ssDNA. The inclusion of high-throughput sequencing techniques has therefore allowed rapid identification of theranostic aptamers from an large randomized library of ssDNA sequences. Our ongoing experiments are focused on coupling of the ovarian cancer cell-specific ssDNA aptamers to contrast agents or cytotoxic drugs. The ssDNA aptamers coupled to contrast agents are specifically being investigated for in vivo imaging of ovarian cancer masses in the peritoneum whereas the aptamers coupled to drugs can be used for the treatment of ovarian cancer.

Citation Format: Rebecca J. Whelan, Arvinder Kapur, Mildred Felder, Jamie Shallcross, Jeff Nie, Manish S. Patankar. Selection of DNA aptamers for an ovarian cancer cell line using high-throughput sequencing [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-THER-1429.
Abstract POSTER-THER-1440: Targeted delivery of doxorubicin loaded nanobins to ovarian cancer cells through the urokinase plasminogen activator system
Authors: Zhang Yilin , Swindell Elden P. , Hankins Patrick L. , Kenny Hilary A. , O'Halloran Thomas V. , Mazar Andrew P. , Lengyel Ernst
Published: August 2015

The therapeutic efficacy of antitumor drugs is often limited by non-specific, systematic delivery. Here we report a novel targeted delivery platform designed by coupling liposomal nanobins (NB) with the urokinase plasminogen activator receptor (u-PAR) antibody (ATN-658). The urokinase system is overexpressed in epithelial ovarian cancer (OvCa) cells and is expressed only at low levels in normal cells. Doxorubicin (Dox), a FDA approved antitumor drug, had been loaded into the nanobins as a payload. The size and surface charge of the nanobins were optimized to facilitate specific binding to u-PAR expressing OvCa cells. Confocal and transmission electron microscopy showed that ATN-658-NB(Dox) was internalized in OvCa cells in a receptor-dependent manner and was released from endosomes as a function of time. This uptake could be blocked by stably down-regulating u-PAR expression in the OvCa cells using shRNA. In an orthotopic ovarian cancer model, athymic mice treated with ATN-658-NB(Dox) had a significantly greater reduction in tumor burden (0.06±0.01g versus 0.1±0.01g; p<0.001) than mice treated with NB(Dox), which was corroborated by in vivo fluorescence imaging results. A survival study showed that targeted nanobins prolonged the survival of mice by 3 days (43 days versus 40 days, median survival) as compared to untargeted nanobins. Immunoblot analysis of tumor sections showed that targeted nanobins induced apoptosis (cleaved caspase 3) and inhibited proliferation (Ki-67) more efficiently when compared to untargeted nanobins. Furthermore, tumor-targeted delivery of ATN-658-ND(Dox) was confirmed by ex vivo fluorescence imaging, FACS analysis, and confocal microscopy. Taken together, our findings show that receptor-mediated delivery of targeted nanobins to cancer cells could serve as a novel approach to ovarian cancer treatment.

Citation Format: Yilin Zhang, Elden P. Swindell, Patrick L. Hankins, Hilary A. Kenny, Thomas V. O’Halloran, Andrew P. Mazar, Ernst Lengyel. Targeted delivery of doxorubicin loaded nanobins to ovarian cancer cells through the urokinase plasminogen activator system [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-THER-1440.
EGFR Kinase Domain Duplication (EGFR-KDD) Is a Novel Oncogenic Driver in Lung Cancer That Is Clinically Responsive to Afatinib
Authors: Gallant Jean-Nicolas , Sheehan Jonathan H. , Shaver Timothy M. , Bailey Mark , Lipson Doron , Chandramohan Raghu , Brewer Monica Red , York Sally J. , Kris Mark G. , Pietenpol Jennifer A. , Ladanyi Marc , Miller Vincent A. , Ali Siraj M. , Meiler Jens , Lovly Christine M.
Published: August 2015

Oncogenic EGFR mutations are found in 10% to 35% of lung adenocarcinomas. Such mutations, which present most commonly as small in-frame deletions in exon 19 or point mutations in exon 21 (L858R), confer sensitivity to EGFR tyrosine kinase inhibitors (TKI). In analyzing the tumor from a 33-year-old male never-smoker, we identified a novel EGFR alteration in lung cancer: EGFR exon 18–25 kinase domain duplication (EGFR-KDD). Through analysis of a larger cohort of tumor samples, we detected additional cases of EGFR-KDD in lung, brain, and other cancers. In vitro, EGFR-KDD is constitutively active, and computational modeling provides potential mechanistic support for its auto-activation. EGFR-KDD–transformed cells are sensitive to EGFR TKIs and, consistent with these in vitro findings, the index patient had a partial response to the EGFR TKI afatinib. The patient eventually progressed, at which time resequencing revealed an EGFR-dependent mechanism of acquired resistance to afatinib, thereby validating EGFR-KDD as a driver alteration and therapeutic target.

Significance: We identified oncogenic and drug-sensitive EGFR-KDD that is recurrent in lung, brain, and soft-tissue cancers and documented that a patient with metastatic lung adenocarcinoma harboring the EGFR-KDD derived significant antitumor response from treatment with the EGFR inhibitor afatinib. Findings from these studies will be immediately translatable, as there are already several approved EGFR inhibitors in clinical use. Cancer Discov; 5(11); 1155–63. ©2015 AACR.

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The Relationship of Sleep Duration with Obesity and Sarcopenia in Community-Dwelling Older Adults
Authors: Chien Meng-Yueh , Wang Li-Ying , Chen Hsi-Chung
Published: August 2015

Background: Numerous studies have reported the relationship between sleep duration and obesity in elderly adults; however, little is known about the relationship of sleep duration and sarcopenia. Objective: We examined the relationship of sleep duration with obesity and sarcopenia in community-dwelling older adults. Methods: A total of 488 community-dwelling adults (224 men and 264 women) aged ≥65 years were included in the analysis. Self-reported sleep duration and anthropometric data were collected. Skeletal muscle mass was estimated using the predicted equation from a bioelectrical impedance analysis measurement. Obesity and sarcopenia were defined according to the body mass index and the skeletal muscle mass index, respectively. Results: The association between sleep duration and sarcopenia exhibited a U shape in older adults. Compared to adults with 6-8 h of sleep, adults with <6 h of sleep had a nearly 3-fold increased likelihood of sarcopenia (odds ratio, OR: 2.76, 95% confidence interval, CI: 1.28-5.96), while adults with ≥8 h of sleep had a nearly 2-fold increased risk of sarcopenia (OR: 1.89, 95% CI: 1.01-3.54). Older adults with a sleep duration <6 h were more prone to obesity (OR: 2.15, 95% CI: 1.08−4.30). After gender stratification, the association between obesity and short sleep duration was more robust in women. Conclusion: There were significant associations of sleep duration with either obesity or sarcopenia in community-dwelling older adults. Gender differences in these associations were also observed.
DCP-LA Activates Cytosolic PKCε by Interacting with the Phosphatidylserine Binding/Associating Sites Arg50 and Ile89 in the C2-Like Domain
Authors: Kanno Takeshi , Tsuchiya Ayako , Shimizu Tadashi , Mabuchi Miyuki , Tanaka Akito , Nishizaki Tomoyuki
Published: August 2015

Background/Aims: The linoleic acid derivative DCP-LA selectively and directly activates PKCε. The present study aimed at understanding the mechanism of DCP-LA-induced PKCε activation. Methods: Point mutation in the C2-like domain on PKCε was carried out, and each kinase activity was monitored in PC-12 cells using a föerster resonance energy transfer (FRET) probe with cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) at the N- and C-terminal ends of PKCε, respectively, or in the cell-free systems using a reversed phase high-performance liquid chromatography (HPLC). Intracellular PKCε mobilization was monitored in PC-12 cells using mRuby-conjugated PKCε. DCP-LA binding to PKCε was assayed using a fluorescein conjugated to DCP-LA at the carboxyl-terminal end (Fluo-DCP). Uptake of DCP-LA into cells was measured in PC-12 ells. Results: In the FRET analysis, DCP-LA decreased the ratio of YFP signal intensity/CFP signal intensity in PC-12 cells and in the cell-free kinase assay, DCP-LA increased area of phosphorylated PKC substrate peptide, indicating DCP-LA-induced PKCε activation. These effects were significantly suppressed by replacing Arg50 and Ile89 by Ala or Asn in the C2-like domain of PKCε. In the fluorescent cytochemistry, DCP-LA did not affect intracellular PKCε distribution. In the cell-free binding assay, Fluo-DCP, that had no effect on the potential for PKCε activation, bound to PKCε, and the binding was inhibited only by mutating Ile89. Extracellularly applied DCP-LA was taken up into cells in a concentration-dependent manner. Although no activation was obtained in the cell-free kinase assay, the broad PKC activator PMA activated PKCε in PC-12 cells in association with translocation towards the cell surface, which was inhibited by mutating I89A. Conclusion: Unlike PMA DCP-LA activates cytosolic PKCε by binding to the phosphatidylserine binding/associating sites Arg50 and Ile89, possibly at the carboxyl-terminal end and the cyclopropane rings, respectively.
Aggravated Liver Injury but Attenuated Inflammation in PTPRO-Deficient Mice Following LPS/D-GaIN Induced Fulminant Hepatitis
Authors: Wang Xiaochen , Yan Shushan , Xu Donghua , Li Jun , Xie Yu , Hou Jiajie , Jiang Runqiu , Zhang Chuanyong , Sun Beicheng
Published: August 2015

Background/Aims: Critical roles of PTPRO and TLR4 have been implicated in hepatocellular carcinoma. However, little is known about their modifying effects on inflammation-related diseases in liver, particularly fulminant hepatitis (FH). We aim to investigate the potential role of PTPRO and its interaction with TLR4 in LPS/D-GaIN induced FH. Methods: A LPS/D-GaIN induced mouse FH model was used. RAW264.7 cells were transfected with PTPRO over-expressed lentiviral plasmids for further investigation. Results: The mortality of PTPRO KO mice is higher than WT mice after LPS/D-GaIN administration. Aggravated liver injury was demonstrated by increased level of serous ALT and AST and numerous hepatic cells death in PTPRO KO mice following LPS/D-GaIN administration. Interestingly, inflammation was attenuated in PTPRO-deficient mice following LPS/D-GaIN administration, which was suggested by decreased inflammatory cytokines (TNF-a, IFN-γ, IL-1ß, IL-6, IL-17A and IL-12) and cells infiltrating into spleen (CD3+IFN-γ+ cells, CD3+TNF-a+ cells, F4/80+/TLR4+ cells). A feedback regulation between PTPRO and TLR4 dependent on NF-γB signaling pathway was demonstrated in vivo and in vitro. Conclusion: PTPRO plays an important role in FH by interacting with TLR4. The crosstalk between PTPRO and TLR4 is a novel bridge linking innate immune and adaptive immune in acute liver injury.
Vasculopathy of Aging and the Revised Cardiovascular Continuum
Authors: Kim Su-A , Park Jeong Bae , O''Rourke Michael F.
Published: August 2015

There have been attempts to explain the process of developments in overt cardiovascular disease, resulting in the presentation of the classic cardiovascular disease continuum and the aging cardiovascular continuum. Although the starting points of these two continua are different, they meet in the midstream of the cycle and reach a consensus at the end of the process. The announcement of the aging cardiovascular continuum made both continua complete, explaining the cardiovascular events in patients without atherosclerotic cardiovascular disease with aging. Impairment of the vascular structure by pulse wave and reflected wave is considered the cause of aortic damage, which influences the development of ischemic heart disease and the development of overt renal disease or cerebrovascular disease. The pathophysiology of vascular aging through pulse wave and its effect on other organs was discussed with Prof. Michael F. O'Rourke who devised the aging cardiovascular continuum.
Effect of Model Variables on in vitro Erosion
Authors: Mistry Miten , Zhu Shanshan , Moazzez Rebecca , Donaldson Nora , Bartlett David W.
Published: August 2015

The aim of this study was to investigate the effects of tooth type (molar/premolar), tooth surface (buccal/lingual), smear layer or no smear layer, storage of specimens in deionised water, mode and speed of agitation, and rinsing method between cycles on mean step height loss and Knoop microhardness (KHN) change. Polished human enamel specimens embedded in acrylic resin were prepared from sound permanent molar and premolar tooth surfaces. A 0.3% (pH 3.2) solution of citric acid was used to erode the specimens, in a cycling procedure, consisting of 10 min immersion followed by rinsing in deionised water for 30 s with a spray bottle, for 5 cycles. The specimens were analysed with a non-contact white light profilometer and KHN. Molar teeth (148.99 ± 24.49 KHN) and buccal surfaces (155.62 ± 30.35 KHN) produced significantly less microhardness change compared to premolar (186.40 ± 20.74 KHN) and lingual surfaces (179.76 ± 23.21 KHN; p < 0001). The effect of storage and rinsing showed little difference in mean step height loss (<1 µm) and microhardness change. With no smear layer a significantly lower mean step height loss and microhardness change (p < 0.001) was observed. Agitation was performed with Orbital, Gyro and See-Saw rockers at 30, 40, 60, and 70 rpm. The mean step height loss was largest for See-Saw at 70 rpm (11.73 ± 0.91 μm) and lowest for Orbital at 30 rpm (2.76 ± 1.12 μm). A statistical difference was found between all types of agitation and speeds (p < 0.001). In conclusion, this study has shown that the variables investigated here have a significant impact on the measurable outcome, highlighting the importance of accurate and detailed method sections.
Calcitonin Gene-Related Peptide Receptor Blocker Inhibits Spontaneous Activity of Human Ureter
Authors: Jankovic Slobodan M. , Jankovic Snezana V. , Stojadinovic Dobrivoje , Stojadinovic Miroslav , Djuric Janko M. , Stojic Isidora
Published: August 2015

Calcitonin gene-related peptide (CGRP) is present in nerve fibers that innervate the human ureter and may have important influence on the motility of this organ. The aim of our study was to investigate whether CGRP could affect the motility of an isolated human ureter. The tension and intraluminal pressure of the isolated ureteral segments were recorded and registered on a personal computer. Both phasic and tonic contractions of the isolated preparations were measured as area under the tension or pressure recordings. CGRP and CGRP fragment 8-37 were separately added to the organ baths in a cumulative way, thereby gradually increasing their concentration in the baths' solution. Alpha-CGRP did not affect either phasic, spontaneous activity or tone of isolated ureteral segments, as measured by both tension and intraluminal pressure. On the other hand, CGRP 8-37 caused concentration-dependent inhibition of spontaneous contractions of the isolated ureteral segments.
Cytoplasmatic and Nuclear YAP1 and pYAP1 Staining in Urothelial Bladder Cancer
Authors: Latz Stefan , Umbach Tine , Goltz Diane , Kristiansen Glen , Müller Stephan C. , Ellinger Jörg
Published: August 2015

Introduction: Yes-associated protein 1 (YAP1), the nuclear effector of the Hippo pathway, plays an important role in many tumor entities. We evaluated staining and clinical significance of YAP1 and phosphorylated YAP1 (pYAP1) in urothelial bladder cancer (BCA). Materials and Methods: We used a tissue micorarray with samples of patients with muscle-invasive bladder cancer (MIBC, n = 192), non-muscle-invasive bladder cancer (NMIBC, n = 192) and normal urothelial bladder tissue (CTRL, n = 38) to determine the immunhistochemical staining of YAP1 and pYAP1. Cytoplasmatic and nuclear levels were evaluated. The t test was used for comparative analysis. Overall survival and progression-free survival were evaluated by Kaplan-Meier estimates and the Cox proportional hazard regression model. Results: Nuclear YAP1 as well as cytoplasmatic pYAP1 levels were higher in CTRL than in BCA, whereby both - NMIBC and MIBC - had lower levels than CTRL. Among patients with MIBC, cytoplasmatic YAP1 and pYAP1 staining decreased with advanced stage. YAP1 and pYAP1 staining did not correlate with the recurrence rate, progression-free, cancer-specific or overall survival. Conclusions: Immunhistochemical staining and subcellular localization of YAP1 and pYAP1 are different for BCA, NMIBC, MIBC and CTRL, indicating that the Hippo pathway is involved in urothelial carcinogenesis.