bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2130: Antitumor activity of marin natural product derived Psammaplin A analogs in human lung cancer cells
Authors: Byun Woong sub , Shin Yoonho , Lee Sang Kook , Park Hyeung-geun , Hong Suckchang
Published: July 2017

Natural products are important sources for bioactive molecules, which have been developed to treat various human health-related symptoms. In our continuos efforts to search for anticancer agents from natural sources, Psammaplin A (PsA), isolated from marine natural products, was found to be a potential candidate in the growth inhibition of cancer cells. PsA, a unique symmetrical bromotyrosine contained in a number of marine sponges of the order Verongida, has exhibited a variety of bioactivities including antimicrobial activity, cytotoxicity against the leukemia cell line, and inhibitory activity of DNA gyrase and DNA topoisomerase. PsA is also known to be a potent inhibitor of DNA methyltransferase and histone deacetylase with growth-inhibitory activity of cancer cells. However, its underlying mechanism of action and the structure-activity relationship (SAR) with PsA analogs have not been elucidated yet. In the present study, twenty-eight synthetic analogs of PsA were newly synthesized and examined the potential of cytotoxicities against cancer cells. A SAR study revealed that the presence of free oxime and disulfide functional groups was responsible for high cytotoxicity. Furthermore, the bromotyrosine component in PsA was relatively tolerable and hydrophobic aromatic groups preserved the cytotoxicity. The aromatic groups were dependent on their size and spatial geometry. Among them, a β-naphthyl derivative of PsA showed a potential cytotoxicity and was comparable to that of PsA. The compound also exhibited a potential antitumor activity in a nude mouse xenograft model. These findings indicated that free oxime and disulfide linker in the chemical structure play an important role for cytotoxicity and PsA analogs might be provided as potential antitumor agents. This work was supported by the National Reseach Foundation of Korea (NRF) Grant funded by the Korean Government (MEST) (NRF-2016M3A9B6903499) and also supported by a National Research Foundation of Korea (NRF) grant funded by the Korean Government (MEST) (No.2009-0083533).

Citation Format: Woong sub Byun, Yoonho Shin, Sang Kook Lee, Hyeung-geun Park, Suckchang Hong. Antitumor activity of marin natural product derived Psammaplin A analogs in human lung cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2130. doi:10.1158/1538-7445.AM2017-2130
Abstract 2148: Phenyl butyrate inhibits pyruvate dehydrogenase kinase 1 and contributes to its anti-cancer effects
Authors: Zhang Wen , Tam Kin Yip
Published: July 2017

In cancer cells, the metabolic features are significantly different from those of normal ones, which are hallmarks of several malignancies. Recent studies brought atypical cellular metabolism, such as aerobic glycolysis or the Warburg effect, into the scientific limelight. Pyruvate dehydrogenase kinase 1 (PDK1), a key enzyme in the pathway of glucose metabolism, could inactivate the pyruvate dehydrogenase (PDH) by phosphorylating it and preserving the substrates pyruvate, lactate and alanine for gluconeogenesis. Overexpression of PDK1 could block the oxidative decarboxylation of pyruvate to satisfy high oxygen demand in cancer cells, while inhibition of PDK1 could upregulate the activity of PDH and rectify the balance between the demand and supply of oxygen, which could lead to cancer cell death. Thus, inhibitors targeting PDK1 might be a promising strategy for cancer treatment by acting on glycolytic tumors while showing minimal side effects on the oxidative healthy organs.

Phenyl butyrate (PB) has been proved to decrease PDH phosphorylation level and increase PDH activity by inhibiting PDK1 in fibroblast cells, PDH deficiency zebrafish and wild type mice. PB has also shown efficacy in many cancers and so far, all of its anti-tumor activity has been attributed to the histone deacetylase (HDAC) inhibitor mode of action. As PDK1/PDH controls the critical switch between oxidative phosphorylation and glycolysis in cancer cells, PDK1 is a key target in tumor metabolism for anti-cancer treatment. We hypothesize that the therapeutic effects of PB in cancers might depend on suppressing PDKs and promoting PDH activity, in addition to its proposed role as HDAC inhibitor. We showed that PB directly inhibited the kinase activity of PDK1 in a purified system. In several different cancer cell lines, PB reduced the phosphorylation level of PDH, increased the mitochondrial respiration, decreased glycolysis in cytoplasm, reversed mitochondrial hyperpolarization, activated some proteins in apoptotic signalling pathway and then induced the apoptosis of cells. In summary, this is the first study indicated that PB could exert its anti-cancer effects through inhibiting PDK1, altering the mitochondrial bioenergetics and inducing apoptosis.

Note: This abstract was not presented at the meeting.

Citation Format: Wen Zhang, Kin Yip Tam. Phenyl butyrate inhibits pyruvate dehydrogenase kinase 1 and contributes to its anti-cancer effects [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2148. doi:10.1158/1538-7445.AM2017-2148
Abstract 2150: Oncogenic role of GAEC1 and its potential modulation with p53 in pathogenesis of colon cancer
Authors: Wahab Riajul , Islam Farhadul , Gopalan Vinod , Lam Alfred King-Yin
Published: July 2017

Introduction: GAEC1 (Gene amplified in esophageal cancer 1), is frequently amplified and overexpressed in colon cancer tissues. In the present study, we aimed to unveil the oncogenic potential of GAEC1 in carcinogenesis of colon cancer by studying the underlying cellular functions and molecular interactions by in vitro and in vivo experiments.

Method: Transient overexpression of GAEC1 with pcDNA3.1-GAEC1 and silencing with GAEC1-siRNA was performed and several downstream assays were done such as migration, clonogenic and apoptotic assay. Analysis of cell kinetics was done using flow cytometry and cell counting kit-8 was used for cell proliferation assay. Immunofluorescence and Western blot assay were used to determine the expression of different target proteins. Co-immunoprecipitation was used to confirm the protein-protein interaction. For xenotransplantation the severely combined immunodeficient (SCID) mice (4 groups, 6 in each group) were injected subcutaneously with GAEC1 shRNA and control shRNA transfected (stable) SW480 and SW48 colon cancer cells.

Result: The overexpression of GAEC1 increased cell proliferation, migration, reduced apoptosis in colon cancer cells. Also, these cells showed cell cycle arrest at the synthetic phase, activation of Bcl-2, K-ras, pAKT proteins as well as inhibition of p53, PUMA, p21 and BAX proteins. Conversely, knockdown of GAEC1 reduced cell proliferation, migration, decreased the phosphorylation of AKT, and induced apoptosis, G2/M phase arrest and cleavage of poly (ADP-ribose) polymerase (PARP). Co-immunoprecipitation revealed GAEC1’s interaction with p53. In addition ectopic over expression and silencing of GAEC1 lead to reciprocal effects of p53 protein expression. Moreover, knockdown of GAEC1 reduced the nuclear translocation of murine double minute 2 protein (mdm2) indicating that GAEC1 is responsible for the degradation of p53 through the direct interaction between mdm2-p53 in nucleus. Furthermore, our in vivo data demonstrate that the loss of GAEC1 inhibits the tumor formation in xenograft model.

Conclusion: Collectively our study demonstrates that GAEC1 exhibits the oncogenic role in colon cancer by reducing expression of p53 through protein-protein interaction which leads to the inhibition of PUMA, p21, BAX, and activation of Bcl-2, K-ras and pAKT protein expression.

Note: This abstract was not presented at the meeting.

Citation Format: Riajul Wahab, Farhadul Islam, Vinod Gopalan, Alfred King-Yin Lam. Oncogenic role of GAEC1 and its potential modulation with p53 in pathogenesis of colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2150. doi:10.1158/1538-7445.AM2017-2150
Abstract 2152: Protein phosphatase Mg2+ and Mn2+ dependent 1F promotes smoking-induced breast cancer by inactivating phosphorylated p53-induced signals
Authors: Chen Li-Ching
Published: July 2017

Previously we demonstrated that the activation of α9-nicotinic acetylcholine receptor (α9-nAchR) signaling by smoking promotes breast cancer formation. To investigate the downstream signaling molecules involved in α9-nAChR-induced breast tumorigenesis, we used real-time polymerase chain reactions and Western blotting to assess expression of protein phosphatase Mg2+/Mn2+ dependent 1F (PPM1F), a Ser/Thr protein phosphatase, in human breast cancer samples (n=167). Additionally, stable PPM1F-knockdown and overexpressing cell lines were established to evaluate the function of PPM1F. The phosphatase activity of PPM1F in nicotine-treated cells was assessed through Western blotting, confocal microscopy, and fluorescence resonance energy transfer. Higher levels of PPM1F were detected in the breast cancer tissues of heavy smokers (n=7, 12.8-fold) greater than of non-smokers (n= 28, 6.3-fold) (**p=0.01). In vitro, nicotine induced PPM1F expression, whereas α9-nAChR knockdown reduced the protein expression of PPM1F. A series of biochemical experiments using nicotine-treated cells suggested that the dephosphorylation of p53 (Ser-20) and BAX (Ser-184) by PPM1F is a critical posttranslational modification, as observed in breast cancer patients who were heavy smokers. These observations indicate that PPM1F may be a mediator downstream of α9-nAChR that activates smoking-induced carcinogenic signals. Thus, PPM1F expression could be used for prognostic diagnosis or inhibited

for cancer prevention and therapy.

Citation Format: Li-Ching Chen. Protein phosphatase Mg2+ and Mn2+ dependent 1F promotes smoking-induced breast cancer by inactivating phosphorylated p53-induced signals [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2152. doi:10.1158/1538-7445.AM2017-2152
Abstract 2156: NSC59984 induces mutant p53 degradation via activating ERK2 pathway-MDM2 axis
Authors: Zhang Shengliang , Zhou Lanlan , Dicker David , El-Deiry Wafik S.
Published: July 2017

Mutant p53 protein is highly expressed in most cancer cells due to its protein stabilization. Mutant p53 loses wild-type p53 function but gains new oncogenic functions in driving tumor growth and drug resistance. Depletion of mutant p53 is an attractive strategy for cancer therapy. We reported a small-molecule NSC59984 destabilizes mutant p53 protein via protein degradation (Zhang et al., Cancer Research, 2015). In this study, we demonstrate that the ERK2 pathway plays a specific role in NSC59984-induced mutant p53 degradation via MDM2-mediated ubiquitination. We observe a sustained-phosphorylation of ERK1/2 in cancer cells treated with NSC59984. ERK1/2 pathway is negatively regulated by MKP-1. We find that MKP-1 activity is partially inhibited by NSC59984 in a dose dependent manner in vitro. These results suggest that NSC5998 sustains ERK1/2 phosphorylation partially via disruption of the feedback-loop between MKP-1 and ERK2 pathway. Knockdown of ERK2 rescues mutant p53 from NSC59984-mediated degradation, and inhibits NSC59984-restoration of p53 signaling in mutant p53-expressing cells. On the contrary, the effect of NSC59984 on the mutant p53 is not inhibited by the blockage of JNK and P38, two pathways negatively regulated by MKP-1. These results suggest that the ERK2 phosphorylation is specifically required for NSC59984-mediated mutant p53 degradation. We further find that NSC59984 induces ERK2-dependent MDM2 phosphorylation. The effect of NSC59984 on MDM2 phosphoryation is partially inhibited by the blockage of ERK2 signaling (via U0126 treatment or Knockdown of ERK2), not by the attenuation of the JNK pathway. Furthermore, NSC59984-mediated mutant p53 protein degradation is inhibited by MDM2 knockdown, and enhanced by MDM2 overexpression in cancer cells. NSC59984-increased ubiquitination of mutant p53 is attenuated by U0126. Our results suggest that the ERK2 signaling pathway-MDM2 axis is a major determinant of NSC59984-mediated mutant p53 degradation. We further demonstrate that NSC59984 induces cell death via apoptosis, and NSC59984-induced cell death is mainly rescued by inhibition of the ERK2 pathway in p53 mutant cancer cells. Taken together, our results suggest that phosphorylated-ERK2 is an important factor required for NSC59984-mediated mutant p53 degradation which may further contribute to NSC59984-induced cell death in cancer cells.

Citation Format: Shengliang Zhang, Lanlan Zhou, David Dicker, Wafik S. El-Deiry. NSC59984 induces mutant p53 degradation via activating ERK2 pathway-MDM2 axis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2156. doi:10.1158/1538-7445.AM2017-2156
Abstract 2165: Deletion of survivin sensitize human hepatocellular carcinoma cells to low dose of doxorubicin and induce apoptosis
Authors: George Joseph , Hayashi Nobuhiko , Saito Takashi , Ozaki Kazuaki , Toshikuni Nobuyuki , Tsuchishima Mutsumi , Tsutsumi Mikihiro
Published: July 2017

Background and Aims: Hepatocellular carcinoma (HCC) is one of the most common cancers in the world and patients with advanced HCC face a dismal prognosis due to lack of effective therapy. Survivin, a member of the family of inhibitor of apoptosis proteins, is highly upregulated in HCC as well as in experimentally induced intrahepatic tumors. Doxorubicin, the only known chemotherapeutic agent for HCC, is cardiotoxic in addition to several well known side effects.

Methods: Survivin gene deletion was established in HepG2, Hep3B, and PLC/PRF/5 human HCC cells using CRISPR/Cas9 system. All the three HCC cells in culture were treated with doxorubicin at various concentrations before and after survivin gene knockout upto 72 hr. TUNEL assay and FACS analysis were performed to demonstrate the induction of apoptosis after doxorubicin treatment. Western blotting was carried out for cleaved fragments of caspase-9 and caspase-3 as well as major apoptotic executioner molecules.

Results: Cell viability studies depicted around 20% cell death at 24 h, 50% at 48 h, and 80% at 72 h after treatment with doxorubicin at 1 µM (final concentration in the media). Among the three cell lines studied, Hep3B cells were more susceptible to doxorubicin compared to HepG2 and PLC/PRF/5 cells. After deletion of survivin gene, the dosage of doxorubicin could reduce to five fold (200 nM, final concentration in the media) with the same cytotoxic effect before the knockout of survivin gene. HCC cells treated with reduced doses of doxorubicin depicted induction of apoptosis that was proved with TUNEL assay and FACS analysis as wells as increased levels of cleaved caspases and major apoptotic executioner molecules.

Conclusions: Our studies demonstrated that blocking of survivin molecule with effective methods would be a successful approach to treat primary hepatic tumors with low and safe doses of doxorubicin and other anticancer agents.

Note: This abstract was not presented at the meeting.

Citation Format: Joseph George, Nobuhiko Hayashi, Takashi Saito, Kazuaki Ozaki, Nobuyuki Toshikuni, Mutsumi Tsuchishima, Mikihiro Tsutsumi. Deletion of survivin sensitize human hepatocellular carcinoma cells to low dose of doxorubicin and induce apoptosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2165. doi:10.1158/1538-7445.AM2017-2165
Abstract 2168: Nucleic acid clamp-mediated recognition and transcriptional modulation of MYC oncogenes through the stabilization of G-quadruplexes
Authors: Hao Taisen , Brooks Tracy A.
Published: July 2017

G-quadruplexes (G4s) tend to cluster around biologically important regions such as promoters of DNA, 5’-UTR of mRNA and telomeres. Modulating the stability of G4 as in these regulatory sequences is an emerging approach for cancer treatment. MYC proto-oncogene is overexpressed in over 80% of tumor types, contributing to deregulated cancer cell proliferation. Silencing MYC expression has been demonstrated to be an effective approach to inhibit tumor growth. Among all the silencing strategies, stabilizing MYC promoter G4 that serves as an on-off switch for the transcription of MYC has been explored by the targeting of small molecules. However, the binding specificity of small molecule retarded the drug development process. Exploring novel MYC G4-targeting strategies is necessary to potentiate the pharmacological specificity of MYC G4 modulation. Here, we adopted a nucleic acid clamp based approach to recognize and stabilize the physiologically predominant MYC G4. Previously, a clamp (clamp A) was been demonstrated to recognize the physiologically relevant MYC G4 with high specificity and to downregulate MYC promoter activity. In the current research, we modified and optimized this clamp to allow for flexibility with labeling and monitoring techniques. The original 18 Å polyethylene glycol phosphate linker connecting the 5’ and 3’ regions of clamp A were substituted with thymine bases of varying lengths; 5 thymines were found to be optimal. The binding specificity and MYC G4 recognizing ability of this optimized clamp A T5 were confirmed by EMSA and ECD; the G4 stabilizing ability of clamp A T5 was demonstrated by DMS footprinting. The cytotoxicity of clamp A T5 was examined by MTS assay on HEK-293 and MCF-7 cells. The intracellular localization of clamp A T5 is being determined microscopically with a 6-FAM-labeled clamp A T5 after being transfected into and incubated with HEK-293 and MCF-7 cells for 48 hr. Nuclear localization is being examined by co-staining with DAPI, and MYC promoter localization is being determined by clustering with a MYC FISH probe. The modification of the clamp is both cost-effective, and enables the detailed study of intracellular functions. This clamp has potential as both a diagnostic tool to inform the use of MYC G4-targeted small molecules, and as a nanotherapeutic.

Citation Format: Taisen Hao, Tracy A. Brooks. Nucleic acid clamp-mediated recognition and transcriptional modulation of MYC oncogenes through the stabilization of G-quadruplexes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2168. doi:10.1158/1538-7445.AM2017-2168
Abstract 2171: Multi-CDK inhibition efficiently suppresses AR function and cell growth in prostate cancer
Authors: Simons Brian W. , Ybanez Maria , Antonarakis Emmanuel S. , Luo Jun , Nelkin Barry D.
Published: July 2017

Metastatic castration resistant prostate cancer (CRPC) continues to result in over 26,000 deaths per year in the United States. For 70 years, therapy for advanced prostate cancer has relied on AR-directed therapies. Unfortunately, even with the second generation androgen deprivation compounds, treated cancers commonly progress to castration resistance and lethality. This progression to castration resistance remains the central therapeutic roadblock for advanced prostate cancer. Importantly, in the vast majority of CRPC cases, the tumor still depends on AR signaling, but this signaling becomes androgen independent. The most common mechanisms, including mutation, protein truncation, and alternative splicing, affect the C-terminal domain, suggesting that antagonism of AR function via the N-terminal domain may be a promising target for CRPC therapy. Several groups have shown that CDKs 1, 5 and 9 activate AR by phosphorylation of the N-terminal domain. Here, we show that the multi-CDK inhibitor roniciclib (BAY1000394) inhibits activation of AR in a panel of prostate cancer cell lines, including several cell lines which exhibit androgen-independent AR signaling via AR-V7 alternative splicing. In vivo, roniciclib inhibited growth of prostate cancer xenografts. Since AR inhibition in prostate cancer has been shown to induce compensatory PI3K signaling, we also treated xenografts with a combination of roniciclib and the PI3K inhibitor copanlisib (BAY 80-6946). This combination was especially effective, and was well tolerated by the mice. Our results suggest that a combination of a multi-CDK inhibitor and a PI3K inhibitor may be promising for CRPC therapy.

Citation Format: Brian W. Simons, Maria Ybanez, Emmanuel S. Antonarakis, Jun Luo, Barry D. Nelkin. Multi-CDK inhibition efficiently suppresses AR function and cell growth in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2171. doi:10.1158/1538-7445.AM2017-2171
Abstract 218: Functional proteomic analysis of TβRI immunocomplex in TGF-β signaling
Authors: Tang Liuya , Meng Zhaojing , Heller Mary , Zhou Ming , Zhang Ying
Published: July 2017

Transforming growth factor-beta (TGF-β) regulates a wide array of biological responses ranging from proliferation to apoptosis, and alterations in its signaling pathway are associated with a variety of human diseases, including cancer. During the activation of TGF-β signaling, TGF-β receptor I (TβRI) plays an important role in transducing signals to downstream effectors. To identify TβRI interacting proteins which are responsible for Smad-dependent pathways as well as Smad-independent pathways, we employed a large-scale quantitative proteomic analysis of TβRI immunocomplex. AML12 cells were labeled with L-Lysine and L-Arginine (K0R0, light) or L-Lysine-13C6-15N2 and L-Arginine-U-13C6-15N4 (K8R10, heavy). The cells cultured in K8R10 medium (H: Heavy) were transfected with Flag-TβRI, while the cells cultured in K0R0 medium (L: Light) were transfected with Flag vector. The cell lysates were combined at equal protein amount, and TβRI immunocomplex were purified using FLAG M2 agarose. The resulting peptides from eluted TβRI immunocomplex were subjected to mass spectrometry quantitation. If a protein whose ratio of H/L was not smaller than 1.3, it was categorized as a constitutively interacting protein of TβRI. A total of 687 proteins were quantitated and 325 proteins were defined as constitutively interacting proteins. Pathway enrichment analysis found these proteins are involved in a number of signaling pathways, which indicates the cross-talks between TGF-β signaling and other signaling. Future work will include validation of interaction between TβRI and candidate proteins, and understand functional significance of candidate proteins in TGF-β signaling or signaling cross-talks by a series of molecular biological approaches.

Citation Format: Liuya Tang, Zhaojing Meng, Mary Heller, Ming Zhou, Ying Zhang. Functional proteomic analysis of TβRI immunocomplex in TGF-β signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 218. doi:10.1158/1538-7445.AM2017-218
Abstract 2191: Multiple imaging reporter labeled acid-degradable dextran nanopolymer as a COX-2 siRNA carrier for COX-2 specific downregulation
Authors: Chen Zhihang , Krishnamachary Balaji , Penet Marie-France , Bhujwalla Zaver M.
Published: July 2017

Cyclooxygenase-2 (COX-2) as a target has attracted interest for multiple degenerative diseases and cancers. COX-2 siRNA provides specific and effective down-regulation of COX-2. However, because artificial cationic polymers can induce proinflammatory signals that increase COX-2 expression, there are few if any reports about the use of artificial cationic polymers as siRNA carrier to downregulate COX-2 in cancer cells. Here we developed a method to efficiently synthesize a multiple imaging reporter labeled biodegradable dextran to use as an efficient cationic polymer carrier for COX-2 siRNA delivery. Amine function groups were conjugated to the dextran platform through acetal bonds. Acetal bonds were broken at acidic conditions that occur in cancer and endocytosis compartments. Rhodamine was labeled to the amine groups to detect degradation and removal of these groups from the cell, while the dextran scaffold was labeled with Cy5.5. The rapid cleavage and release of amine groups minimized the proinflammatory side effects of the positively charged amine groups.Colorimetric assay of Cy5.5 and rhodamine in different pH buffer indicated that the amine group was cleaved at pH 5.5 buffer but was stable in pH 7.4 buffer. Fluorescence imaging showed that the dextran siRNA nanoplex entered the cells through endocytosis that provided acidic conditions for the breaking of the amine group. For the first time, the intracellular breaking of acetal bonds was clearly visualized by multiple imaging reporters. Because the rapid cleavage and release of amine groups minimized the proinflammatory side-effects, quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay indicated that this dextran COX-2 siRNA nanoplex could downregulate COX-2 expression efficiently. Prostaglandin E2 level decreased significantly with COX-2 siRNA/dextran treatment. In vivo imaging demonstrated that the COX-2 siRNA/dextran nanoplex accumulated in MDA-MB-231 tumors. QRT-PCR and western blot assays of COX-2 levels in tumor tissue demonstrated that this nanoplex significantly downregulated COX-2 expression in vivo efficiently and within 24h. This dextran nanopolymer can be used as a safe, reproducible, and biocompatible siRNA carrier to effectively reduce COX-2 expression in cancer cells and tumors.

Citation Format: Zhihang Chen, Balaji Krishnamachary, Marie-France Penet, Zaver M. Bhujwalla. Multiple imaging reporter labeled acid-degradable dextran nanopolymer as a COX-2 siRNA carrier for COX-2 specific downregulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2191. doi:10.1158/1538-7445.AM2017-2191
Abstract 22: MM-161, a first-in-class pan-FGFR antibody
Authors: Dake Tamara , Finn Greg , Geddie Melissa , Kohli Neeraj , Razlog Maja , Xu Lihui , Paragas Violette , Yuzugullu Haluk , Ghassemifar Sara , Hashambhoy-Ramsay Yasmin , McDonagh Charlotte , Muda Marco , Schoeberl Birgit
Published: July 2017

Aberrant signaling of the FGFR pathway has long been known to promote tumorigenesis and angiogenesis across multiple cancer indications. However, the development of an effective and well-tolerated FGFR targeted inhibitor has been hindered by the need to block the activation of multiple mitogenic receptors while avoiding significant toxicities associated with blocking endocrine FGF ligands.

Here we disclose for the first time a novel FGFR targeted antibody, MM-161, designed to block ligand-dependent signaling driven by all four FGF receptors, specifically the IIIc-isoforms. MM-161 is well tolerated in mice and cynomolgus monkeys with no significant weight loss observed in either species. Efficacy studies demonstrated that MM-161 monotherapy leads to significant tumor growth inhibition or tumor regression of xenografts of human lung, renal and endometrial cancer amongst others. Importantly, MM-161 has a dual mechanism of action by inhibiting both proliferation and angiogenesis. We will present data illustrating that inhibition of multiple FGFRs is desirable to achieve tumor regression. Furthermore, we will show combination studies with relevant standard of care therapies in models of lung and renal cancer.

Taken together, our preclinical data strongly supports the clinical evaluation of MM-161 in cancer patients.

Citation Format: Tamara Dake, Greg Finn, Melissa Geddie, Neeraj Kohli, Maja Razlog, Lihui Xu, Violette Paragas, Haluk Yuzugullu, Sara Ghassemifar, Yasmin Hashambhoy-Ramsay, Charlotte McDonagh, Marco Muda, Birgit Schoeberl. MM-161, a first-in-class pan-FGFR antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 22. doi:10.1158/1538-7445.AM2017-22
Abstract 220: Proteomic identification of therapeutics targets for Enzalutamide resistance in Castration Resistant Prostate Cancer
Authors: VELOT Lauriane , Levesque Dominique , Boisvert François-Michel , Bisson Nicolas , Pouliot Frédéric
Published: July 2017

Prostate cancer (PC) is the most frequently diagnosed cancer in Canadian men and is the 3rd cause of cancer mortality. The Androgen Receptor (AR) is activated by androgens (e.g. testosterone), which leads to prostatic cell proliferation. The primary treatment for advanced PC is androgen-deprivation therapy, which is achieved via surgical or chemical castration. Nevertheless, most PC will become castration-resistant (CRPC). Although new anti-androgens (e.g. Enzalutamide) were developed to improve patients’ survival, their efficacy is still very limited and most CRPC patients will die from the disease within a few years. We postulate that by gaining insights into AR signalling networks in the context of CRPC, we could better direct patients towards the best-suited therapy and propose new therapeutic targets.

To this aim, we took advantage of an innovative proteomics approach, namely proximity-labeling (BioID), to characterize global AR signalling networks in hormone-dependant LAPC4 cells. We identified 45 AR-associated proteins in non-stimulated cells, 35 of which were not previously reported. Upon androgenic stimulation, the AR signalling network increased to 320 proteins, including 278 (253 were novel) that were restricted to androgen-stimulated cells. Enzalutamide treatment resulted in a loss of 259 proteins from the network when compared to stimulated cells. As expected, this reproduced quite faithfully the status of non-stimulated LAPC4 cells. Interestingly, we identified 4 proteins in the AR network specifically after Enzalutamide treatment. These are interesting targets that may be relevant for the acquisition or the prediction of Enzalutamide resistance. Hence, they could become in time alternative therapeutic targets for CRPC treatment, to be used when Enzalutamide fails.

Citation Format: Lauriane VELOT, Dominique Levesque, François-Michel Boisvert, Nicolas Bisson, Frédéric Pouliot. Proteomic identification of therapeutics targets for Enzalutamide resistance in Castration Resistant Prostate Cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 220. doi:10.1158/1538-7445.AM2017-220
Abstract 2208: Peptide-mediated 'miniprep' isolation of extracellular vesicles is suitable for high-throughput proteomics; method evaluation and application in colon cancer
Authors: Wit Meike De , Knol Jaco , Reus Inge de , Schelfhorst Tim Tim , Bishop-Currey Logan , Grieken Nicole van , Piersma Sander , Pham Thang V. , Fijneman Remond J. , Meijer Gerrit A. , Verheul Henk , Jimenez Connie R.
Published: July 2017

Objective:

Extracellular vesicles (EVs) are cell-secreted membrane vesicles enclosed by a lipid bilayer derived from endosomes or from the plasma membrane. Since they are released into body fluids, and their cargo includes tissue-specific and disease-related molecules, EVs represent a rich source for disease biomarkers. However, standard ultracentrifugation methods for EV isolation (UC-EV) are laborious, time-consuming, and require high inputs.

Method:

A recently described isolation method, which can be performed at small ‘miniprep’ scale, utilizes specific Heat Shock Protein (HSP)-binding peptides to aggregate HSP-decorated EVs (Ghosh et al. (2014), PLoS ONE 9:e110443). The authors showed comparable results for their method (abbreviated HSP-EV here) and UC-EV, but a detailed proteomic comparison was lacking. Therefore, we compared both methods using label-free proteomics of replicate EV isolations from HT-29 cancer cell-conditioned medium. Subsequently we applied this technique on secretomes of fresh human colorectal cancer (CRC) (n=17) and colon adenoma (n=4) tissue as well as patient-matched normal colon tissue.

Results:

Despite a 30-fold different input scale (UC-EV: 60 ml versus HSP-EV: 2 ml), both methods yielded comparable numbers of identified proteins (3115 versus 3085), with reproducible identifications (72.5% versus 75.5%) and spectral count-based quantification (average CV 31% versus 27%). EVs obtained by either method contained established EV markers and proteins linked to vesicle-related gene ontologies. In the EV fraction of the tissue secretomes 6390 proteins were identified, of which 471 proteins were significantly 5-fold more present in CRC samples than in normal tissue EVs. Gene ontology analysis revealed enrichment of nuclear proteins involved in DNA damage response, chromosome organization and RNA processing in the CRC EVs.

Conclusions:

The HSP-EV method provides an advantageous, simple and rapid approach for EV isolation from small amounts of biological samples, enabling high-throughput analysis in a biomarker discovery setting.

Citation Format: Meike De Wit, Jaco Knol, Inge de Reus, Tim Tim Schelfhorst, Logan Bishop-Currey, Nicole van Grieken, Sander Piersma, Thang V. Pham, Remond J. Fijneman, Gerrit A. Meijer, Henk Verheul, Connie R. Jimenez. Peptide-mediated 'miniprep' isolation of extracellular vesicles is suitable for high-throughput proteomics; method evaluation and application in colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2208. doi:10.1158/1538-7445.AM2017-2208
Abstract 2210: Scavenging for lethal prostate cancer biomarkers in FFPE tissue
Authors: Ramberg Håkon , Braadland Peder R. , Stensland Maria E. , Rakaee Mehrdad , Nygård Ståle , Øgren Olov , Berge Viktor , Svindland Aud , Richardsson Elin , Souza Gustavo A. de , Andersen Sigve , Taskén Kristin A.
Published: July 2017

The aim of this study was to identify protein biomarkers that can be used to stratify indolent and aggressive prostate cancer using formalin-fixed paraffin-embedded tissue (FFPE) from radical prostatectomy specimens. The protein expression profiles of benign and malign samples from 23 patients, who had either died of prostate cancer or were alive 10 years after diagnosis, were determined using an Easy nLC1000 Nano-LC system connected to a quadrupole-Orbitrap (Q Exactive) mass spectrometer equipped with a nanoelectrospray ion source. The generated MS raw files were submitted to MaxQuant software for protein identification. Differential expression of LFQ-normalized intensities was tested by Cox proportional hazards modeling and LASSO (least absolute shrinkage and selection operator) regression analysis within the R software environment. The label free Orbitrap analysis identified 3523 unique proteins from the FFPE tissue samples. LASSO of protein expression profiles of indolent and aggressive prostate cancer tissue samples identified ten proteins which could stratify the groups. Seven of the proteins were significantly correlated with time to prostate cancer specific mortality in Cox proportional hazards modeling (p-value < 0.05). The selected proteins were found to be linked to NFkappaB signaling, cell structure, translation and metabolic pathways. Finally, proteins capable of distinguishing benign from malignant prostate tissue were identified by LASSO, and 22 of these were significantly different between the groups (Bonferroni-adjusted p-value < 0.05). Selected candidate biomarkers are currently being validated by immunohistochemical analyses of 535 radical prostatectomy specimens from two independent Norwegian cohorts with long follow-up time.

Citation Format: Håkon Ramberg, Peder R. Braadland, Maria E. Stensland, Mehrdad Rakaee, Ståle Nygård, Olov Øgren, Viktor Berge, Aud Svindland, Elin Richardsson, Gustavo A. de Souza, Sigve Andersen, Kristin A. Taskén. Scavenging for lethal prostate cancer biomarkers in FFPE tissue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2210. doi:10.1158/1538-7445.AM2017-2210
Abstract 2214: Adaptive dynamic artificial polyligand targeting (ADAPT): a method to identify exosomal proteins from a prostate cancer cell line
Authors: Hornung Tassilo , Logie Stephen , Bondre Aniket S. , Maher Varun , Richards Melissa N. , Zarkovic Jelena , Tinder Teresa T. , O'Neill Heather A. , Miglarese Mark R. , Spetzler David B.
Published: July 2017

In the recent years it was demonstrated that a multitude of body fluids contains substantial amounts of exosomes, extracellular vesicles with sizes ranging between 40 and 100 nm. Those vesicles have protein profiles characteristic of their cells of origin. It was shown that exosomes play a role in cell-to-cell communication making them attractive targets to identify early disease stage biomarkers. Cancer heterogeneity is known for a long time to be an important clinical determinant of patient outcome. We developed the highly multiplexed ADAPT platform to capture systems-based biological signatures that may reflect the molecular heterogeneity of various cancer types and help to improve diagnosis of the disease. Exosomes from two prostate cancer cell lines, VCaP and LNCaP, were used to train ssDNA libraries to discriminate them. A highly diverse library of 1012 oligonucleotides (ODNs) was subjected to five rounds of positive and negative selection against exosomes from VCaP and LNCaP prostate cancer cell lines. Individual ODNs that bound preferably to exosomes from VCaP cells were identified by NGS, resynthesized and binding of co-precipitated ODNs to VCaP exosomes was verified by qPCR. LC-MS/MS was used to identify binding partners of ODNs bound to VCaP exosomes. Several of those binding partners (CHMP1b/2a/4b, VPS28, Syntenin-1) were found to be part of the ESCRT machinery, which participates in exosomes biogenesis. It was found that those proteins are overexpressed in human cancers. In addition, we identified the chemokine I-TAC, which is overexpressed in blood and tissue of men with advanced prostate adenocarcinomas. Finally, we found hnRNP-1, a cancer associated splicing factor, and the cold shock proteins RNPL and A18 hnRNP. Knock-down of these cold shock proteins has been shown to enhance chemotherapeutic cell killing of prostate cells. ADAPT is an unbiased profiling platform that identifies cancer associated proteins expressed on exosomes. This platform can be deployed against multiple cancer types and offers broad potential applications in biomarker discovery.

Citation Format: Tassilo Hornung, Stephen Logie, Aniket S. Bondre, Varun Maher, Melissa N. Richards, Jelena Zarkovic, Teresa T. Tinder, Heather A. O'Neill, Mark R. Miglarese, David B. Spetzler. Adaptive dynamic artificial polyligand targeting (ADAPT): a method to identify exosomal proteins from a prostate cancer cell line [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2214. doi:10.1158/1538-7445.AM2017-2214
Abstract 2225: Green tea polyphenols suppress tumor growth and invasion by targeting matrix metalloproteinases, RECK and TIMP-3, in a mouse model implanted with prostate tumors
Authors: Shankar Eswar , Bhaskaran Natarajan , Kanwal Rajnee , Gupta Sanjay
Published: July 2017

Green tea polyphenols (GTP) and its major constituent, epigallocatechin-3-gallate (EGCG) reactivate epigenetically silenced genes in cancer cells that reduces invasiveness and migration capabilities; however, the mechanisms whereby these effects occur are not well understood. RECK, a novel tumor suppressor and the tissue inhibitor of matrix metalloproteinase-3 (TIMP-3) genes negatively regulates matrix metalloproteinases (MMPs) and inhibits tumor invasion, angiogenesis and metastasis. In the present study, we demonstrate that GTP mediate epigenetic induction of RECK and TIMP-3 thereby playing a key role in suppressing invasiveness and gelatinolytic activity of MMP-2 and MMP-9 in vivo in an orthotopic implantation model of human prostate cancer. Athymic nude mice were implanted with human prostate cancer LNCaP tumor in the ventral prostate for 2 weeks and later treated DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (AZA), histone deacetylase inhibitor, trichostatin A (TSA) and histone methyltransferase inhibitor, 3-Deazaneplanocin A (DZNep) individually at 0.1 mg/kg body weight intraperitoneally at alternate days/week; and combination of AZA+TSA and DZNep+TSA at similar doses and times; whereas GTP was provided peroral by gavage at 7.5 and 15.0 mg/kg body weight freshly prepared in 100µl PBS. Treatment of mice with GTP resulted in marked decrease in tumor growth and its local invasion in dose dependent manner, compared to treatment with epigenetic inhibitors and their combination after 8 weeks of intervention. GTP treatment significantly reduced serum levels of MMP-2, MMP-9 and VEGF, compared to treatment with epigenetic inhibitors alone. Combination of AZA+TSA exhibited similar effect which was equivalent to the lower dose of GTP treatment. Furthermore, GTP treatment significantly reduced EZH2 and H3K27me3 and class I HDAC protein levels in tumors. GTP partially reversed the hypermethylation status of RECK and TIMP3 gene and significantly enhanced their protein expression in the tumor tissue. Inhibition of VEGF, MMP-2 and MMP-9 levels were also noted after GTP treatment in tumor tissue in dose-dependent manner. Our findings suggest that induction of RECK and TIMP-3 are key epigenetic events modulated by GTP that results in suppression of matrix degradation and angiogenesis to delay prostate cancer invasion and its subsequent progression.

Citation Format: Eswar Shankar, Natarajan Bhaskaran, Rajnee Kanwal, Sanjay Gupta. Green tea polyphenols suppress tumor growth and invasion by targeting matrix metalloproteinases, RECK and TIMP-3, in a mouse model implanted with prostate tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2225. doi:10.1158/1538-7445.AM2017-2225
Abstract 2227: Fgf2 from visceral adipose tissue stimulates neoplastic transformation of nonmalignant epithelial cells
Authors: Chakraborty Debrup , Benham Vanessa , Demireva Elena Y. , Bullard Blair , Bernard Jamie J.
Published: July 2017

Background: Adiposity plays a crucial role in the pathogenesis and prognosis of different types of cancers. Epidemiological evidence suggests visceral adipose tissue (VAT) and high-fat diets (HFD) are associated with increased cancer risk however the mechanism is not understood. The aim of this study was to explore the factors in VAT that stimulate neoplastic transformation.

Methods: We modeled visceral adiposity-stimulated neoplastic transformation using our novel ex vivo system of VAT-condition medium stimulated epithelial cell transformation (measured by growth in soft agar) and our in vivo murine lipectomy model of ultraviolet light B (UVB)-induced, VAT promoted skin tumor formation. FgfR1(-/-) stable cells were generated by using CRISPR-Cas9 technology and were used to investigate the role of fibroblast growth factor-2 (FGF2) and FGFR1 dependent signaling in neoplastic transformation, both in vitro and in vivo.

Results: Only the VAT of obese mice fed a HFD [not VAT from low-fat diet (LFD) fed mice] stimulated neoplastic transformation of skin epithelial cells. Furthermore, human VAT stimulated both skin and mammary epithelial cell transformation. The differences in VAT activity between LFD and HFD fed mice and human donors were associated with the levels of FGF2. Circulating levels of FGF2 were associated with non-melanoma tumor formation in vivo. Human and mouse VAT failed to stimulate transformation in FgfR1(-/-) cells and do not form tumors when injected in Nude mice in vivo.

Conclusion: Collectively, our data show FGF2 released from VAT and its interaction with FGFR1 is a novel and potential direct path of VAT-enhanced tumorigenesis. Blocking the FGFR1 induced signaling in VAT of abdominally obese individuals may be an important cancer prevention strategy as well as an adjuvant therapy for improving outcomes following cancer diagnosis.

Citation Format: Debrup Chakraborty, Vanessa Benham, Elena Y. Demireva, Blair Bullard, Jamie J. Bernard. Fgf2 from visceral adipose tissue stimulates neoplastic transformation of nonmalignant epithelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2227. doi:10.1158/1538-7445.AM2017-2227
Abstract 2232: Dietary flavonoids, CYP1A1 genetic variants, and the risk of colorectal cancer
Authors: Kim Jeongseon , Cho Young Ae , Lee Jeonghee , Oh Jae Hwan , Sohn Dae Kyung , Chang Hee Jin
Published: July 2017

The role of dietary flavonoid intake in colorectal carcinogenesis might differ according to flavonoid subclasses and individual genetic variants related to carcinogen metabolism. Therefore, we examined whether greater habitual dietary intake of flavonoid subclasses was associated with a lower risk of colorectal cancer and whether CYP1A1 genetic variants altered this association. A semi-quantitative food frequency questionnaire was used to assess the dietary intake of six flavonoid subclasses (flavonols, flavonones, flavanones, flavan-3-ols, anthocyanidins, and isoflavones) in 923 patients with colorectal cancer and 1,846 controls; furthermore, CYP1A1 genetic variants (rs4646903 and rs1048943) were genotyped. A logistic regression was used to investigate dietary flavonoid intake, CYP1A1 genetic variants, and their interaction in relation to colorectal cancer risk. Among the subclasses of flavonoids, flavonols (OR [95% CI] = 0.10 [0.06, 0.16], highest vs. lowest quartile) and flavan-3-ols (OR [95% CI] = 0.54 [0.41, 0.71], highest vs. lowest quartile) were significantly associated with colorectal cancer risk after adjusting for confounders. Homozygous variants of CYP1A1 rs4646903 showed a protective effect on the risk of rectal cancer (OR [95% CI] = 0.60 [0.37, 0.96], CC vs. TT/TC). Carriers of the CC homozygous variant with high flavonol intake showed a greater decrease in colorectal cancer risk compared with T allele carriers with low flavonol intake (P for interaction = 0.02), particularly regarding rectal cancer (P for interaction = 0.005). In conclusion, the effect of dietary flavonoid intake on colorectal cancer risk differs by flavonoid subclasses and CYP1A1 genetic variants.

Citation Format: Jeongseon Kim, Young Ae Cho, Jeonghee Lee, Jae Hwan Oh, Dae Kyung Sohn, Hee Jin Chang. Dietary flavonoids, CYP1A1 genetic variants, and the risk of colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2232. doi:10.1158/1538-7445.AM2017-2232