bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5669: Design and synthesis of membrane type matrix metalloproteinase (MT-MMP) targeted anti-tumor agents.
Authors: Falconer Robert A. , Youssef Ahmed M. , Gimenez Warren Javier , Morais Goreti R. , Mitchell Andrew , Shnyder Steve D. , Gill Jason H. , Patterson Laurence H. , Loadman Paul M.
Published: August 2014

Design and synthesis of membrane type matrix metalloproteinase (MT-MMP) targeted anti-tumor agents

Membrane-type matrix metalloproteinases (MT-MMPs) are highly active in tumors, but absent or inactive in normal tissues. MT-MMPs are known to be elevated in the majority of human tumors and to be central to tumor invasion and angiogenesis. Our objective has been to design inactive prodrugs that are converted to a potent drug by selected MMPs within the tumor microenvironment.

We report the design and synthesis of a series of peptide-based conjugates of azademethylcolchicine (a potent colchicine analogue) designed to be selectively cleaved by MT-MMPs in the tumor microenvironment. Azademethylcolchicine was attached to the peptide C-terminus, while the N-terminus is protected from non-specific cleavage by exopeptidases through a masking group. A series of N-terminal masking groups, including unnatural amino acids, substituted aromatic groups, alkyl spacers and more complex endcaps was employed. Insights have been gained into the MT-MMP peptide recognition sequence and the relative importance of individual amino acids with regards MMP-selectivity. The P2’ position in particular has been extensively studied through substitution by both natural amino acid and unnatural amino acids.

We have investigated the efficiency of hydrolytic cleavage of these conjugates to release the active agent in both tumor and normal tissues. Results indicate significant effects of P2’ amino acid substitution upon stability and conversely activation in tumor and normal tissues (liver, kidney, lung, plasma), with half-lives of hydrolysis ranging from 2-60 min in tumor and 3-300 min in normal tissues.

In conclusion, a series of peptide-based prodrug conjugates of azademethylcolchicine was synthesized. Hydrolytic activation of these prodrugs in tumor tissue was rapid compared to liver and other normal tissues. From this screen, ICT-2588, which includes tyrosine at the P2’ position, emerged as the agent with a hydrolytic cleavage rate most suited for further evaluation as a tumour activated peptide-based conjugate.

Citation Format: Robert A. Falconer, Ahmed M. Youssef, Javier Gimenez Warren, Goreti R. Morais, Andrew Mitchell, Steve D. Shnyder, Jason H. Gill, Laurence H. Patterson, Paul M. Loadman. Design and synthesis of membrane type matrix metalloproteinase (MT-MMP) targeted anti-tumor agents. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5669. doi:10.1158/1538-7445.AM2013-5669
Abstract 567: Dual functions of Mad1 in mitosis and DNA damage response.
Authors: Yang Chunying , Hao Jianwei , Kong Dejuan , Xu Bo
Published: August 2014

The spindle assembly checkpoint (SAC) and the DNA damage checkpoint are two evolutionarily conserved mechanisms by which the fidelity of chromosome segregation in response to various DNA lesions is protected. These checkpoints are believed to function independently. However, there is growing evidence showing that proteins involved in the two systems have overlapping functions. Here we show that the mitotic arrest-deficient protein 1 (Mad1), a critical component of the SAC, is also required for the DNA damage response (DDR). We found that depletion of Mad1 led to cellular hypersensitivity to ionizing radiation (IR) and prolonged persistence of the H2AX foci. Interestingly, we observed Mad1 to be hyperphosphorylated both in mitosis and in response to DNA damage induced by IR in an ATM (Ataxia Telangiectasia Mutated) dependent manner. ATM is an essential DDR kinase which is also activated in mitosis and required for the SAC. We found that ATM phosphorylated Mad1 on Serine 214 both in vitro and in-cell. We further demonstrated that ATM-mediated Mad1 Serine 214 phosphorylation was required for both SAC and optimal DDR. Collectively, these findings reveal the dual function of ATM-mediated Mad1 phosphorylation in mitosis and DDR.

Citation Format: Chunying Yang, Jianwei Hao, Dejuan Kong, Bo Xu. Dual functions of Mad1 in mitosis and DNA damage response. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 567. doi:10.1158/1538-7445.AM2013-567
Abstract 5680: Identification and characterization of a novel class of ATX inhibitors.
Authors: Fells James , Lee Sue Chin , Norman Derek , Fujiwara Yuko , Tsukahara Ryoko , Liu Jianxiong , Kirby Jason , Nelson Sandra , Tigyi Gabor
Published: August 2014

The lysophospholipase D (autotaxin, ATX) plays an important role in cancer invasion and metastasis. A High-throughput screen of over 10,000 compounds from the University of Cincinnati Drug Discovery Center, identified several small-molecule inhibitors with IC50 values ranging from nanomolar to low micromolar. The pharmacology of the two most potent compounds, 966791(1) and 918103(2), were further characterized in enzyme, cellular, and whole animal systems. Both of these compounds, significantly reduced cell invasion in melanoma cancer cell lines without any apparent cytotoxicity. Compound (1) was shown to be a competitive inhibitor of ATX for both FS-3 and Pnp-TMP substrates, while compound (2) was competitive only for FS-3. This suggest that compound (2) targets the hydrophobic pocket not the active site of ATX, which was supported by docking of compounds 1 and 2, along with mutagenesis data. The potency of the compounds was comparable to each other in each of the assays. Current ATX inhibitors reported in literature target the active site in the catalytic domain. In our search for ATX inhibitors we identified compounds able to inhibit both the active site and hydrophobic pocket. We conclude that these data suggest compounds solely inhibiting the hydrophobic pocket are sufficient to disrupt the ATX-LPA signaling axis can serve as specific small-molecule modulators for mechanistic studies of ATX as well as a potential leads for the development of anti-cancer drugs.

Citation Format: James Fells, Sue Chin Lee, Derek Norman, Yuko Fujiwara, Ryoko Tsukahara, Jianxiong Liu, Jason Kirby, Sandra Nelson, Gabor Tigyi. Identification and characterization of a novel class of ATX inhibitors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5680. doi:10.1158/1538-7445.AM2013-5680
Abstract 5681: Modulation of Wnt/β-catenin signaling by niclosamide and related derivatives.
Authors: Mook Robert A. , Chen Minyong , Lu Jiuyi , Barak Larry S. , Lyerly H. Kim , Chen Wei
Published: August 2014

The Wnt signal transduction pathway is dysregulated in many cancers, including colorectal cancer (CRC) where activating mutations in this pathway occur in over 80% of sporadic colorectal cancers. As a result, drugs that inhibit the pathway are highly sought-after as the basis of a new generation of innovative therapeutic agents. Unfortunately, a gap exists in the ability to target the pathway with small, drug-like molecules, a gap that is the barrier to discovering drugs targeting defects in this pathway. Recently, we reported the FDA-approved anthelmintic drug niclosamide inhibits Wnt/β-catenin signaling via a novel mechanism that internalizes Frizzled receptors and leads to decreases in Dishevelled and β-Catenin proteins, even in cells harboring mutations in APC and β-catenin. The biological target driving inhibition of Wnt signaling by niclosamide is unknown. To support efforts to translate these findings in clinical trials and assist target identification efforts, we interrogated the mechanism and structure-activity relationships. We will present our findings indicating inhibition of Wnt signaling by niclosamide appears unique among the structurally-related anthelmintic agents tested and that Wnt/β-catenin activity was dependent on small changes in the chemical structure of niclosamide - including the ability to convert inhibition to activation. Overall, the findings support efforts to identify the target of niclosamide inhibition of Wnt/β-catenin signaling and the discovery of novel inhibitors of Wnt/β-catenin signaling to treat cancer, and thus accelerate the translational application of niclosamide and its derivatives to improve patient survival from cancer.

Citation Format: Robert A. Mook, Minyong Chen, Jiuyi Lu, Larry S. Barak, H. Kim Lyerly, Wei Chen. Modulation of Wnt/β-catenin signaling by niclosamide and related derivatives. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5681. doi:10.1158/1538-7445.AM2013-5681
Abstract 5682: Design and preclinical evaluation of single agents with combination chemotherapy potential.
Authors: Gangjee Aleem , Pavana Roheeth K. , Ihnat Michael A. , Thorpe Jessica E. , Disch Bryan C. , Bastian Anja , Bailey-Downs Lora C. , Hamel Ernest , Bai Rouli
Published: August 2014

Antiangiogenic agents target tumor mechanisms involved in blood vessel formation vital for tumor growth and metastases. However, these agents are mainly cytostatic and usually not tumoricidal. Several combinations of antiangiogenic and cytotoxic agents have shown significant promise in the clinic, and several clinical trials are in progress. Single agents with both antiangiogenic activities and cytotoxicity would afford agents that would circumvent pharmacokinetic problems of multiple agents, avoid drug-drug interactions, could be used at lower doses to alleviate toxicity, be devoid of overlapping toxicities, and could delay or prevent tumor cell resistance. We designed, synthesized and evaluated a novel compound AG119 that inhibits both vascular endothelial growth factor receptor-2 (VEGFR-2) affording antiangiogenic effects and tubulin for cytotoxic effects in a single agent. Structural modifications of AG119 afforded AG321 that inhibited platelet derived growth factor receptor-β (PDGFR-β) and epidermal growth factor receptor (EGFR) in addition to VEGFR-2 and tubulin. The novel compounds AG119 and AG321 were potent inhibitors (activity comparable to that of combretastatin A-4) of bovine brain tubulin assembly and of [3H]colchicine binding to tubulin, indicating that these compounds bind at the colchicine site on tubulin. Both compounds have potency comparable to those of sunitinib and semaxinib in tumor cell lines overexpressing VEGFR-2. AG321 has potency comparable to that of erlotinib in tumor cell lines overexpressing EGFR and potency comparable to those of erlotinib, DMBI and PD153035 in cell lines overexpressing PDGFR-β. Both AG119 and AG321 also inhibited angiogenesis in the chorioallantoic membrane (CAM) assay. AG321 is a unique compound with four separate mechanisms of action in a single agent including inhibition of tubulin. This is unprecedented and to our knowledge, is the first of its kind. In mice, AG119 reduced tumor size and vascularity in two flank xenograft models [the BLBC MDA-MB-435 model and U251 glioma model] and in a 4T1 triple negative breast orthotopic allograft model. In these in vivo models, the activity of AG119 was superior to those of temozolomide (U251), docetaxel and sunitinib (MDA-MB-435 and 4T1) without overt toxicity to the animals.

Citation Format: Aleem Gangjee, Roheeth K. Pavana, Michael A. Ihnat, Jessica E. Thorpe, Bryan C. Disch, Anja Bastian, Lora C. Bailey-Downs, Ernest Hamel, Rouli Bai. Design and preclinical evaluation of single agents with combination chemotherapy potential. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5682. doi:10.1158/1538-7445.AM2013-5682
Abstract 571: Overactive Cdk2 permits polyploidy by overwhelming the tetraploidy checkpoint.
Authors: Jahn Stephan , Corsino Patrick , Law Mary , Davis Brad , Law Brian
Published: August 2014

The p53-dependent tetraploidy checkpoint serves to block the proliferation of cells that double their genome without dividing. Oftentimes, cancers will contain a tetraploid sub-population, indicating either an ability to bypass this checkpoint or that the checkpoint is no longer functional. In studying the chromosomal instability of tumor cell lines from mice with mammary expression of a constitutively active Cyclin D1-Cdk2 fusion protein (D1K2), we noticed an increased rate of polyploidization in the presence of the spindle-poison paclitaxel. This was recapitulated in an MCF10A cell line model, and it was discovered that this is due to the inability of the tetraploidy checkpoint to completely stop DNA replication in tetraploid cells with constitutively active Cdk2. Biochemical analysis shows that tetraploid cells expressing D1K2 maintain Rb phosphorylation longer than tetraploid control cells, matching the phenotype seen in cells that have p53 stably knocked down. Further DNA replication, yielding octaploid cells, appears to further intensify the tetraploidy checkpoint, arresting the cells. Due to the fact that approximately 50% of all breast cancers overexpress Cyclin D, these observations raise intriguing questions about the treatment of tumors with overactive Cdk2. While it appears these tetraploid MCF10A cells are unviable, we can not account for other mutations that may exist in vivo and allow the proliferation of a chromosomally unstable tetraploid tumor population.

Citation Format: Stephan Jahn, Patrick Corsino, Mary Law, Brad Davis, Brian Law. Overactive Cdk2 permits polyploidy by overwhelming the tetraploidy checkpoint. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 571. doi:10.1158/1538-7445.AM2013-571
Abstract 581: Surfactant-free PLGA encapsulation facilitates delivery of LY294002 to endoplasmic reticulum and enhances its cytotoxicity.
Authors: Hou Chia-Cheng
Published: August 2014

LY294002 (LY) is a potent inhibitor of phosphatidylinositol 3-kinases (PI3Ks); however, bio-applications of LY are limited by its poor solubility and pharmacokinetic profile. Poly(lactic-co-glycolic acid) (PLGA) is a biodegradable nanomaterial that can be used as a carrier for drug delivery. Although LY-loaded polyvinyl alcohol (PVA)-containing PLGA nanoparticles (PVA-LY NPs) have been developed to improve LY solubility, the ratio of NP internalization is unsatisfactory and the cytotoxicity of PVA-LY NPs is inferior to that of free LY. LY-loaded surfactant-free PLGA nanoparticles (SF-LY NPs) were developed to improve the therapeutic efficacy of LY. The size of SF-LY NPs was in the range of 80-100 nm, the shape was spherical and the NPs were monodispersed. The loading capacity of LY in SF NPs was 7.4μg/mg with a sustained-released pattern. In four lung cancer cell lines, SF-LY NPs showed a stronger growth inhibitory effect, sustained suppression of the serine/threonine kinase AKT activation, and increased intracellular uptake of LY compared with PVA-LY NPs or free LY. In addition, it was observed that SF NPs tend to accumulate in the endoplasmic reticulum (ER) and subsequently induce pronounced ER stress. After injection into tumors, SF-LY NPs exhibited a stronger antitumor effect than free LY. These findings indicated that the surfactant-free formulation of PLGA is an ideal vector to carry LY and that SF-LY NPs possess promising anticancer activity.

Citation Format: Chia-Cheng Hou. Surfactant-free PLGA encapsulation facilitates delivery of LY294002 to endoplasmic reticulum and enhances its cytotoxicity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 581. doi:10.1158/1538-7445.AM2013-581
Abstract 586: Genomic and functional characterizations of phosphodiesterase 4Din human cancers.
Authors: Lin Dechen , Xu Liang , Ding Ling-Wen , Sharma Arjun , Liu Li-Zhen , Yang Henry , Tan Patrick , Vadgama Jay , Karlan Beth , Lester Jenny , Urban Nicole , Schummer Michèl , Doan Ngan , Said Jonathan , Walsh Martin , Patel Paresma , Thomas Craig , Chan Daniel , Koeffler Phillip
Published: August 2014

Background

Phosphodiesterase 4D (PDE4D) degrades the secondary messenger adenosine 3’, 5’-cyclic monophosphate (cAMP). PDE4D recently was implicated in cancer cell proliferation, but alterations in its genome and function in tumors are unclear.

Methods

PDE4D gene copy number determined by SNP-CHIP was comprehensively analyzed in a large number of solid tumor specimens from diverse origins (n=5,569). Expression of PDE4D gene transcripts were examined in 971 primary tumors, as well as established cancer cell lines. PDE4D protein expression in 11 different types of cancers were evaluated by immunohistochemistry (n=165). Endogenous PDE4D in multiple types of tumor cell lines was suppressed by both shRNAs and compound 26B, a novel inhibitor of PDE4D, and the effects on apoptosis and proliferation were examined. We further investigated the molecular mechanisms involved. Effect of ectopic expression of PDE4D on tumor growth was assessed both in vitro and in vivo.

Results

Homozygous deletion of PDE4D occurred in 198 cases out of 5,569 primary solid tumors (3.56%), with most of them being internal microdeletions. Unexpectedly, these internal microdeletions did not result in loss of its gene products, as the expression of PDE4D mRNA and protein was not compromised in the PDE4D-microdeleted samples. Immunohistochemistry staining revealed that PDE4D protein levels were up-regulated in several types of tumors. Importantly, genetic and pharmacological suppression of endogenous PDE4D caused apoptosis and growth inhibition preferentially in a variety of cancer cells but not in non-malignant epithelial cells. We further showed that anti-tumor events triggered by PDE4D-suppression were lineage-dependently associated with induction of BIM and down-regulation of MITF. Moreover, ectopic expression of the PDE4D short isoform (PDE4D2) enhanced the proliferation of melanoma both in vitro and in vivo.

Conclusions

We show for the first time that although targeted by genomic homozygous microdeletions, alternatively spliced PDE4D can function as a tumor-promoting factor. PDE4D represents a novel, targetable enzyme of cancer cells.

Citation Format: Dechen Lin, Liang Xu, Ling-Wen Ding, Arjun Sharma, Li-Zhen Liu, Henry Yang, Patrick Tan, Jay Vadgama, Beth Karlan, Jenny Lester, Nicole Urban, Michèl Schummer, Ngan Doan, Jonathan Said, Martin Walsh, Paresma Patel, Craig Thomas, Daniel Chan, Phillip Koeffler. Genomic and functional characterizations of phosphodiesterase 4Din human cancers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 586. doi:10.1158/1538-7445.AM2013-586
Abstract 587: A novel non-COX-inhibitory sulindac derivative induces autophagic cell death by inhibiting Akt signaling in lung cancer cells.
Authors: Gurpinar Evrim , Grizzle William E. , Shacka John J. , Li Nan , Keeton Adam B. , Piazza Gary A.
Published: August 2014

Nonsteroidal anti-inflammatory drugs (NSAIDs) such as sulindac sulfide (SS) have shown promising antineoplastic activity in multiple tumor types, but toxicities resulting from cyclooxygenase (COX) inhibition limit their chronic use for chemoprevention. We recently described a N,N-dimethylethyl amine derivative of SS referred to as sulindac sulfide amide (SSA) that does not inhibit COX-1 or -2, yet displays potent tumor cell growth inhibitory activity. Here, we studied the basis for the growth inhibitory effects of SSA on human lung adenocarcinoma cell lines. SSA potently inhibited the growth of lung tumor cells with IC50 values of 2-5 μM compared with 44-52 μM for SS. SSA also suppressed DNA synthesis and caused a G0/G1 cell cycle arrest more potently than SS. SSA-induced cell death was associated with characteristics of autophagy, including an increase in acidic vesicular organelle content, elevation of the levels of LC3-II, and an increase in autophagic flux. siRNA knockdown of Atg7 attenuated SSA-induced autophagy and cell death. Surface exposure of phosphatidylserine, cleavage of caspases 3 and 7, as well as PARP were detectable with SSA treatment, although the pan-caspase inhibitor, ZVAD was not able to rescue viability. SSA treatment also inhibited Akt/mTOR signaling and the expression of downstream proteins that are regulated by this pathway. Overexpression of a constitutively active form of Akt was able to reduce autophagy markers and confer resistance to SSA-induced cell death. Our findings provide evidence that SSA induces autophagic cell death in lung cancer cell lines by suppressing Akt/mTOR signaling. This unique mechanism of action along with its increased potency and lack of cyclooxygenase inhibition support the development of SSA or related analogs for the prevention and/or treatment of lung cancer. Funding provided by NIH/NCI grants CA131378 and CA148817.

Citation Format: Evrim Gurpinar, William E. Grizzle, John J. Shacka, Nan Li, Adam B. Keeton, Gary A. Piazza. A novel non-COX-inhibitory sulindac derivative induces autophagic cell death by inhibiting Akt signaling in lung cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 587. doi:10.1158/1538-7445.AM2013-587
Abstract 637: Antisense transcripts and R-loops caused by DNA topoisomerase I inhibition by camptothecin at human active CpG island promoters.
Authors: Marinello Jessica , Chillemi Giovanni , Bueno Susana , Manzo Stefano G. , Capranico Giovanni
Published: August 2014

DNA Topoisomerase I (Top1) is specifically inhibited by camptothecin (CPT), a natural product with effective anticancer activity. CPT can stabilize a DNA-Top1 DNA cleavage complex that can lead to irreversible DNA breakage at replication forks, and transcription-dependent genome instability. The bulk of cellular Top1 activity is required at transcribing regions to modulate DNA supercoils generated by elongating RNA polymerases. However, the molecular effects of CPT inhibition of Top1 at transcriptional levels are not fully established. By next generation sequencing analyses of bisulfite-treated RNAs extracted from human HCT116 cancer cells, we here show that CPT promoted high levels of antisense tags specifically at active promoters containing CpG islands (CGI). We found several not annotated transcripts close to Refseq genes, the majority of which were promoter-associated antisense transcripts increased by CPT. We identified 256 and 84 of such transcripts in HCT116 cells and Top1-silenced HCT116-siRNATop1 cells, respectively. We validated CPT-stimulated antisense transcripts by PCR in both cell lines, and found that several of them could form R-loops at corresponding genomic regions by affinity purification with a recombinant inactive mutant RNaseH1. Drug effects were independent from replication, and required both Top1 and ongoing transcription. In addition, CPT induced an immediate burst and a subsequent rapid reduction of Top1-DNA cleavage complexes at active, but not inactive, promoters indicating a partial, time-dependent removal of Top1 from chromatin. Our findings demonstrate that Top1 activity prevents accumulation of antisense R-loops and RNAs at human active CGI promoters by relaxing negative DNA supercoils. The transcriptional CPT effects can contribute to drug therapeutic activity in cancer and other diseases. We will discuss our findings in relation to transcription regulation and genome instability caused by interference with Top1 activity.

Citation Format: Jessica Marinello, Giovanni Chillemi, Susana Bueno, Stefano G. Manzo, Giovanni Capranico. Antisense transcripts and R-loops caused by DNA topoisomerase I inhibition by camptothecin at human active CpG island promoters. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 637. doi:10.1158/1538-7445.AM2013-637
Abstract 638: PBF overexpression causes increased p53 ubiquitination and degradation via MDM2.
Authors: Ryan Gavin , Seed Robert , Read Martin , Fong Jim , Turnell Andrew , Smith Vicki , Franklyn Jayne , McCabe Christopher , Boelaert Kristien
Published: August 2014

The pituitary tumor-transforming gene-binding factor (PBF) is a relatively uncharacterised proto-oncogene, which is overexpressed in thyroid tumours. PBF elicits tumor growth in nude mice, whilst thyroid targeted transgenic overexpression in the PBF-Tg mouse induces hyperplasia and macrofollicular lesions, accompanied by induction of the E2 ubiquitin ligase Rad6. Our previous unpublished data showed that PBF binds to p53, and reduces stimulation of downstream target genes by competitive binding. Further, half-life studies of p53 showed reduced p53 stability when PBF was overexpressed in K1 and TPC-1 thyroid papillary cancer cell lines, and ubiquitination assays confirmed this was due to increased ubiquitination and subsequent degradation by the proteasome. Now, GST pull-down assays demonstrate direct binding between PBF and MDM2, the principal negative regulator of p53. The competitive inhibitor of p53-MDM2 binding, Nutlin-3, revealed that the increased degradation of p53 observed when PBF was overexpressed was mediated by MDM2. No change in p53-MDM2 binding stringency was detected when PBF expression was ameliorated by siRNA treatment, and MDM2 subcellular localisation was unchanged by PBF overexpression. However, co-immunoprecipitation assays revealed that PBF specifically interacts with Rad6, which has previously been shown to regulate p53 ubiquitination. Further, PBF-Tg mice demonstrated significantly induced genetic instability at 6 weeks of age, as determined by FISSR-PCR. Thus we propose that aberrantly expressed PBF functionally inactivates p53 via a complex interplay between Rad6 and MDM2, thus promoting genetic instability and tumorigenesis. Our studies, carried out in a panel of thyroid and colorectal cells lines, suggest this may be pertinent not only to thyroid cancer, but to colorectal and other tumour types.

Citation Format: Gavin Ryan, Robert Seed, Martin Read, Jim Fong, Andrew Turnell, Vicki Smith, Jayne Franklyn, Christopher McCabe, Kristien Boelaert. PBF overexpression causes increased p53 ubiquitination and degradation via MDM2. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 638. doi:10.1158/1538-7445.AM2013-638
Abstract 646: Identification of novel epigenetic biomarkers for platinum-based chemotherapy resistance in high grade serous ovarian cancer.
Authors: Tomar Tushar , Alkema Nicolette G. , De Meyer Tim , van Criekinge Wim , Klip Harry G. , Meersma Gert Jan , van der Zee Ate GJ , de Jong Steven , Wisman G. Bea A.
Published: August 2014

Platinum-based chemotherapy has been standard of care for advanced stage ovarian cancer (OC) patients since the last 3 decades. However, majority of patients relapse within 6 months with drug-resistant disease, which entitle OC among the most lethal gynecological malignancy. Besides ‘’classic’’ clinicopathological features of the primary tumor, no biological parameters have shown clinically predictive and prognostic value for chemoresponse in OC. In the present study, we are focusing on the identification of novel DNA methylation markers in response to platinum-based chemotherapy in high grade serous OC. To accomplish this aim, we performed next generation sequencing on methylation-enriched genomic DNA, isolated from frozen OC patient material from responders (more than 18 months progression free survival) and non-responders (less than 6 months progression free survival) to platinum chemoresponse (n=10 in each group).

Subsequent robust biostatistics and comparative expression data analysis revealed a list of significantly differentially methylated genes of which a number were checked by methylation specific PCR (MSP) on a large platinum sensitive and resistance OC cell line panel. Gene ontology analysis showed that these genes are frequently associated with cell-fate determination, lineage commitment, transcriptional factor binding and ions-transporter system.

Selected candidate methylation markers are currently being validated by pyrosequencing. Moreover, these selected markers are in-silico validated with publically available methylation and expression databases. Next, selected candidate methylation markers will be validated by quantitative-MSP on an independent OC patient cohort. Ultimately, this study will provide a DNA methylation profile for identifying those patients that may benefit from platinum-based chemotherapy in combination with epigenetic treatment modalities.

Citation Format: Tushar Tomar, Nicolette G. Alkema, Tim De Meyer, Wim van Criekinge, Harry G. Klip, Gert Jan Meersma, Ate GJ van der Zee, Steven de Jong, G. Bea A. Wisman. Identification of novel epigenetic biomarkers for platinum-based chemotherapy resistance in high grade serous ovarian cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 646. doi:10.1158/1538-7445.AM2013-646
Abstract 675: Jarid2 is a novel target for lung cancer therapy.
Authors: Hong Young K. , Zhang Mary , Atay Scott , Raiji Manish , Upham Trevor , Hong Julie A. , Rao Mahadev , Schrump David S.
Published: August 2014

Increasing evidence indicates that aberrant expression/activity of polycomb repressive-complex-2 (PRC-2) contributes to the pathogenesis of a variety of human malignancies including lung cancers. In recent studies we have demonstrated that depletion of EZH2- a core component of PRC-2, either by biochemical or pharmacologic techniques inhibits the malignant phenotype of lung cancer cells, indicating that PRC-2 is a target for epigenetic therapy of lung cancer. In the present study, we sought to further examine the potential efficacy of targeting PRC-2 in lung cancers via depletion of Jarid2, which modulates gene expression in stem cells in part via targeting PRC-2 to DNA. Quantitative RT-PCR and immunoblot experiments demonstrated over-expression of Jarid2 in cultured lung cancer cells and primary lung cancers relative to normal respiratory epithelia or histologically normal lung tissues. In subsequent experiments, shRNA techniques were used to inhibit Jarid2 expression in lung cancer cells. Quantitative RT-PCR and immunoblot experiments demonstrated that knock-down of Jarid2 had relatively modest effects on global H3K27Me3 levels in lung cancer cells. These finding were in contrast to knock-down of EZH2, which markedly inhibited H3K27 trimethylation in these cancer cells. Interestingly, knock-down of Jarid2 inhibited in vitro proliferation of lung cancer cells by approximately 70% relative to respective controls, and significantly diminished growth of subcutaneous lung cancer xenografts in athymic nude mice. The anti-proliferative effects of Jarid2 knock-down were relatively comparable to those observed following depletion of EZH2 in lung cancer cells. Jarid2 knock- down only modestly enhanced cytotoxicity mediated by DZNep in lung cancer cells. Microarray experiments are currently in progress to compare gene expression profiles in lung cancer cells mediated by Jarid2 knock-down relative to EZH2 depletion or DZNep treatment, and to delineate the mechanisms by which Jarid2 enhances the malignant phenotype of lung cancer cells. Collectively, these preliminary findings warrant further analysis of Jarid2 expression during pulmonary carcinogenesis, and suggest that Jarid2 may be a novel target for lung cancer therapy.

Citation Format: Young K. Hong, Mary Zhang, Scott Atay, Manish Raiji, Trevor Upham, Julie A. Hong, Mahadev Rao, David S. Schrump. Jarid2 is a novel target for lung cancer therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 675. doi:10.1158/1538-7445.AM2013-675
Abstract 684: Novel potent curcumin analogues EF-31 and UBS109 inhibit DNA methylation and induce SPARC expression in pancreatic cancer.
Authors: Ganji Purnachandra Nagaraju , Zhu Shijun , Mamoru Shoji , El-Rayes Bassel
Published: August 2014

Purpose/Objectives: DNA methylation is an essential step in epigenetic modification and carcinogenesis. HSP90 and NF-κB have a central role in DNA methylation. Curcumin down regulates HSP90 and NF-kB. UBS109 and EF-31 are potent synthetic analogues of curcumin. We tested the hypothesis that EF-31 and UBS-109 can inhibit DNA methylation and induce SPARC through inhibition of NF-kB and HSP90 in pancreatic cancer cell lines.

Methods: Two pancreatic cancer cell lines were used, PANC-1 and Mia-PaCa2. Cells were either untreated (control) or treated with curcumin (20μM), EF-31 (750nM) and UBS-109 (250nM) for 24 hours. Western blot and RT-PCR analyses were carried out to determine the effect of curcumin and its analogues EF-31 and UBS-109 on different signal molecules involved in carcinogenesis. Electromobility shift assay (EMSA) was performed in pancreatic cells to evaluate the effects of ganetespib on NF-kB activity. Immunocyto and histo chemistry assays were also performed.

Results: The treatment of pancreatic cancer cell lines with curcumin and its analog EF-31 and UBS-109 inhibited NF-κB and HSP90 at transcriptional and translational level resulting in inhibition of DNMT1, decrease in CpG island methylation and up-regulation of SPARC. EF-31 and UBS109 were more potent at inhibition of DNMT1 than curcumin. To confirm that the observed effects were mediated by NF-kB, a stable transfection with p65 (NF-kB) was performed. Cells with overexpression of NF-κB resulted in increased DNMT-1, HSP90 protein and decreased SPARC. Inhibition of NF-kB using EF31 or UBS109 resulted in up-regulation of SPARC and inhibition of the DNMT due to NF-κB down-regulation. Similar effects were observed in tumors from animals treated with EF-31 and UBS-109.

Conclusion: UBS109, EF31 and curcumin can inhibit DNA methylation through down-regulation of DNMT-1 expression by NF-kB. Inhibition of methylation was associated with expression of silenced genes such as SPARC. UBS109 and EF31 are significantly more potent than curcumin. UBS109 and EF31 are promising agents for therapy in pancreatic cancer.

Citation Format: Purnachandra Nagaraju Ganji, Shijun Zhu, Shoji Mamoru, Bassel El-Rayes. Novel potent curcumin analogues EF-31 and UBS109 inhibit DNA methylation and induce SPARC expression in pancreatic cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 684. doi:10.1158/1538-7445.AM2013-684
Abstract 691: Methylation of APC and GSTP1 in non-neoplastic tissue adjacent to prostate tumour and mortality from prostate cancer.
Authors: Grasso Chiara Celestina , Richiardi Lorenzo , Fiano Valentina , Zugna Daniela , Gillio Tos Anna , Delsedime Luisa , Merletti Franco
Published: August 2014

Prostate cancer is the most common tumour in men in many populations, with a crude incidence of 120-130 per 105 in Europe and the United States. The incidence of prostate cancer has increased dramatically since the introduction of prostate-specific antigen (PSA) testing at the end of the 1980s; opportunistic PSA-based screening for prostate cancer is widespread, although its ability to decrease mortality is still being debated.

We studied gene methylation in non-neoplastic tissue adjacent to prostate tumours (NTAT) in association with prostate cancer mortality, as a marker to discriminate between indolent and aggressive prostate tumours.

From two cohorts of consecutive prostate cancer patients diagnosed at one pathology ward in Turin, Italy, we selected 157 patients with available NTAT and followed them up for more than 14 years. We obtained DNA from NTAT in paraffin-embedded prostatic tumour tissues and used probe real-time PCR to analyse methylation of the glutathione S-transferase (GSTP1) and adenomatous polyposis coli (APC) gene promoters. Prevalence of APC and GSTP1 methylation in the NTAT was between 40 and 45%. It was associated with methylation in prostatic tumour tissue for the same two genes as well as with high Gleason. The hazard ratio (HR) of prostate cancer mortality was 2.16 (1.13-4.13) for APC methylation, and 2.83 (1.42-5.65) for GSTP1 methylation in NTAT. It changed to 1.75 (0.95-3.22) and 1.61 (0.80-3.22) after adjusting for Gleason score and methylation in prostatic tumour tissue. Comparison of 2 vs. 0 methylated genes in NTAT revealed a HR of 3.84 (1.83-8.03), which decreased to 2.12 (1.03-4.37) after adjustment. Results were stronger in the first 5 years of follow-up (adjusted HR: 3.03, 95% CI: 1.20-7.67) and in the biopsy sub-cohort (HR: 5.91, 95% CI: 1.00-35.0).

This results show that changes in gene methylation are an early event in prostate carcinogenesis and may play a role in cancer progression. Gene methylation in NTAT is a possible prognostic marker.

Citation Format: Chiara Celestina Grasso, Lorenzo Richiardi, Valentina Fiano, Daniela Zugna, Anna Gillio Tos, Luisa Delsedime, Franco Merletti. Methylation of APC and GSTP1 in non-neoplastic tissue adjacent to prostate tumour and mortality from prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 691. doi:10.1158/1538-7445.AM2013-691
Abstract 7: Berg Interrogative Biology™ platform unravels novel biomarkers FLNB and LY9 in combination with PSA enhances specificity in serum of patients with prostate cancer.
Authors: Sarangarajan Rangaprasad , Chan Joyce , Grund Eric , Vishnudas Vivek K. , Luan Shen , Akmaev Viatcheslav R. , Narain Niven R.
Published: August 2014

Prostate Specific Antigen (PSA) test is the predominant screening test for prostate cancer. Utility is limited by the low predictive power to differentiate patients with indolent tumors from aggressive disease with potential for metastasis. Identification of potential biomarkers to increase prediction of patient population with propensity for progression to metastatic disease remains the overarching unmet need in prostate cancer screening. This study reports the identification of a panel of potential biomarkers using the Berg Interrogative Biology™ platform, a novel non-hypothesis driven approach to characterize disease specific molecular signatures for the identification of physiological drivers of disease etiology. Filamin B (FLNB) and lymphocyte antigen 9 (LY9) were two of several markers identified from a high confidence causal network of prostate cancer model using the Berg Interrogative Biology™ platform. Both FLNB and LY9 were validated in a small case-control retrospective study in human serum samples of patients with prostate cancer and age-matched normal controls. The utility of FLNB and LY9 as potential biomarkers in prostate cancer was determined by comparing the serum levels to that of PSA to establish benchmark comparison. Statistically significant elevation of the PSA, FLNB, and LY9 concentrations was detected in the prostate cancer group compared to the age-matched normal control group. Statistical analyses were performed to determine the sensitivity and specificity of the molecular markers by ROC curve analysis. The predictive power of FLNB or LY9 alone was comparable to that of the PSA test. The combination of FLNB and LY9 with PSA out-performed PSA alone with AUC=0.975 in the study. The study provides data in support of FLNB and LY9 as biomarkers with potential to improve screening outcomes when used in combination with PSA. Efforts are currently underway in large patient cohorts to validate the clinical utility of FLNB and LY9 as candidate biomarkers for use in the screening of prostate cancer with correlate to the Gleason score to enable more instructive data to the practitioner.

Citation Format: Rangaprasad Sarangarajan, Joyce Chan, Eric Grund, Vivek K. Vishnudas, Shen Luan, Viatcheslav R. Akmaev, Niven R. Narain. Berg Interrogative Biology™ platform unravels novel biomarkers FLNB and LY9 in combination with PSA enhances specificity in serum of patients with prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 7. doi:10.1158/1538-7445.AM2013-7
Abstract 704: Inhibitors of telomerase and of poly(ADP-Ribose)polymerases synergize to limit the lifespan of pancreatic cancer cells.
Authors: Burchett Katrina M. , Ouellette Michel M.
Published: August 2014

Telomerase is required for the unlimited lifespan of cancer cells. Pancreatic cancers are almost always positive for telomerase activity and inhibiting telomerase in these cells could limit their lifespan. In this project, we have characterized the biology of telomeres in a panel of 11 pancreatic cancer cell lines and have examined the effects of GRN163L (Imetelstat), a potent telomerase inhibitor, on these cells. GRN163L inhibited telomerase in all 11 pancreatic cancer cell lines, with IC50 ranging from 50 nM to 200 nM. Continuous exposure of AsPC-1, CAPAN1 and CD18 cells to 1 μM GRN163L resulted in telomere shortening, induction of crisis, and loss of the cultures. Crisis In these cells was accompanied by activation of a DNA damage response (γ-H2AX) and evidence of both senescence (SA-β-galactosidase) and apoptosis (Floating cells, sub-G1 DNA content, PARP1 cleavage). In L3.6 cells, continuous GRN163L exposure led to the emergence of a GRN163L-resistant sub-population of cells. Over the last year, these cells have been expanded at successively higher concentrations of GRN163L and are still dividing in the presence of 10 μM GRN163L. To shorten telomeres and limit the lifespan of these cells, an alternate approach was to use inhibitors of poly(ADP-ribose)polymerases (PARP) to block the parsylation of TRF1 and TRF2, thereby enhancing the telomerase-inhibitory activity of the Shelterin complex. Our results show that combining GRN163L with the general PARP inhibitor 3-aminobenzamide (3AB) was sufficient to shorten telomeres and limit the lifespan of the GRN163L-L3.6 resistant cells. These experiments were then repeated in the parental L3.6 cells to determine if 3AB could be used to block the development of GRN163L-resistance. Whereas 3AB alone was not sufficient to induce crisis, 3AB did synergize with GRN163L to accelerate the timing of crisis and block the emergence of GRN163L resistant cells. These results raise the possibility that inhibitors of poly(ADP-ribose)polymerases could be exploited in the clinic to accelerate the effects of anti-telomerase therapies.

Citation Format: Katrina M. Burchett, Michel M. Ouellette. Inhibitors of telomerase and of poly(ADP-Ribose)polymerases synergize to limit the lifespan of pancreatic cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 704. doi:10.1158/1538-7445.AM2013-704
Abstract 708: Overcoming the cisplatin resistance of HNSCC cells through Chk1/2 inhibition.
Authors: Gadhikar Mayur A. , Sciuto Maria Rita , Ortega Marcus Vinicus Alves , Pickering Curtis , Monroe Marcus , Osman Abdullah , Neskey David , Sturgis Erich M. , Myers Jeffrey N. , Frederick Mitchell J.
Published: August 2014

Despite the use of multimodality therapy employing cisplatin to treat patients with advanced stage head and neck squamous cell carcinoma (HNSCC), there is an unacceptably high rate of treatment failure. TP53 is the most commonly mutated gene in HNSCC, and the impact of p53 mutation on response to cisplatin treatment is poorly understood. Here we show unambiguously that wild type TP53 (wtp53) is associated with sensitivity of HNSCC cells to cisplatin treatment while mutation or loss of TP53 is associated with cisplatin resistance. We also demonstrate that senescence is the major mode of cell death that accounts for the cisplatin responses in wtp53 HNSCC cells and that cisplatin resistance in p53 null or mutant TP53 cells is due their resistance to senescence induction. Given the dependence on Chk1/2 kinases to mediate the DNA damage response in p53 deficient cells, there is potential to exploit this to therapeutic advantage through targeted inhibition of the Chk1/2 kinases. Treatment of p53 deficient HNSCC cells with the Chk inhibitor leads to sensitization to cisplatin treatment through the induction of mitotic cell death. Therefore, the most common genomic alteration in HNSCC, p53 mutation, can lead to a decreased therapeutic response to cisplatin that can be overcome using a synthetic lethal approach with Chk inhibition. These pre-clinical data provide evidence that a personalized approach to the treatment of HNSCC based on Chk inhibition in p53 mutant tumors may be feasible. The is the first report presenting unambiguous evidence that HNSCC cells respond to cisplatin in a p53 dependent manner. In addition, we clarify that in the presence of wtp53 the major mode of cell death in response to cisplatin in HNSCC cells is senescence, and not apoptosis. TP53 mutations have been identified in 60% of HNSCC cases and many of these patients’ tumors are resistant non-surgical treatment options. By showing dramatic sensitization to cisplatin through addition of Chk1/2 kinase inhibitor in cells with different HNSCC p53 mutations, we present a promising therapeutic approach that warrants further pre-clinical and clinical investigation.

Citation Format: Mayur A. Gadhikar, Maria Rita Sciuto, Marcus Vinicus Alves Ortega, Curtis Pickering, Marcus Monroe, Abdullah Osman, David Neskey, Erich M. Sturgis, Jeffrey N. Myers, Mitchell J. Frederick. Overcoming the cisplatin resistance of HNSCC cells through Chk1/2 inhibition. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 708. doi:10.1158/1538-7445.AM2013-708