bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4542: Regulation of the cell cycle by metformin is p21-dependent in lung cancer
Authors: Templeton Amanda , Ramesh Rajagopal
Published: May 2015

The oral antidiabetic agent metformin has antineoplastic activity that is likely mediated by mechanisms independent of its hypoglycemic effects. Previously reported in human non-small cell lung carcinoma (NSCLC) cell lines, 24 h treatment with metformin induced growth inhibition and G0/G1 phase arrest through induction of the energy sensing kinase AMP-activated kinase (AMPK) and the DNA damage responsive protein ataxia telangectasia mutated (ATM). AMPK and ATM are both known to regulate G0/G1 and G2 phases through modulation of the p53-p21 axis. Therefore, the objective of our study was to elucidate the molecular mechanism of metformin induced cell cycle arrest in NSCLC. Congruent with previous findings, we demonstrate that 5mM metformin treatment for up to 72 h inhibits growth in H1299 and A549 NSCLC cell lines, but not in the non-cancerous fibroblast cell line MRC-9. In H1299 and A549 cells, growth inhibition was associated with an induction of phosphorylation of AMPK and ATM as well as a reduction in phosphorylation of AKT and mTOR. Extending these findings using H1299 cells, metformin consistently induced only a transient G0/G1 phase arrest at 24 h by downregulating cyclin D1 and p27 and upregulating p21 and at 72 h a G2/M-arrest by accumulating cyclin B1 and upregulating p21 and p27. These arrests were associated with ATM and Chk-2 activation and resultant inhibitory phosphorylation of cyclin dependent phosphatase CDC25c. Importantly, p21 knock down in H1299 cells demonstrated the coordinate loss of p27 stabilization, attenuation of the G0/G1 phase arrest at 24 h, and decrease in inhibitory phosphorylation of pan-cyclin dependent kinases. Therefore, we demonstrate for the first time in NSCLC that metformin activates G1 and G2 -checkpoint responses, but the fidelity of the G1-arrest is compromised by the downregulation of p27. However, p21-dependent upregulation of p27 facilitates a G2/M arrest at 72 h. The ability of metformin to control aberrant oncogenic cell-cycle regulation by inducing negative cell-cycle regulators p21 and p27 may provide a novel therapeutic strategy in lung cancer.

Note: This abstract was not presented at the meeting.

Citation Format: Amanda Templeton, Rajagopal Ramesh. Regulation of the cell cycle by metformin is p21-dependent in lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4542. doi:10.1158/1538-7445.AM2014-4542
Abstract 4546: COX-2 inhibitors arrest prostate cancer cell cycle progression by downregulation of kinetochore/centromere proteins
Authors: Bieniek Jared , Childress Chandra , Swatski Matthew , Yang Wannian
Published: May 2015

Epidemiologic studies likewise revealed a decreased incidence of prostate cancer in patients taking non-steroidal anti-inflammatory drugs (NSAIDs) and selective COX-2 inhibitors. Previous in vitro studies have shown that inhibition of cyclooxygenase-2 (COX-2) arrests cancer cell growth. However, the molecular mechanism underlying the growth arrest effect of COX-2 inhibition remains elusive. In this study, we demonstrated that treatment with the COX-2 inhibitors celecoxib and CAY10404 significantly inhibited prostate cancer LNCaP cell proliferation. Flow cytometric analysis confirmed the cell cycle arrested at the G2/M phase and immunofluorescent staining indicated that inhibition of COX-2 prevented the cells from entering into mitosis. Further biochemical analysis showed that inhibition of COX-2 induced a dramatic down-regulation of key proteins in the kinetochore/centromere assembly, such as ZWINT, Cdc20, Ndc80, CENP-A, Bub1 and Plk1. Furthermore, the EP1 receptor antagonist SC51322 produced similar effects to the COX-2 inhibitors on LNCaP cell proliferation and down-regulation of kinetochore/centromere proteins, which did not occur in the EP2, EP3 and EP4 receptor antagonists, suggesting the COX-2 inhibitors arrest the cell proliferation and down-regulate kinetochore/centromere proteins through inactivation of the EP1 receptor. Our studies indicate that inhibition of COX-2 can arrest prostate cancer cell growth through inactivation of the EP1 receptor signaling and down-regulation of kinetochore / centromere proteins.

Citation Format: Jared Bieniek, Chandra Childress, Matthew Swatski, Wannian Yang. COX-2 inhibitors arrest prostate cancer cell cycle progression by downregulation of kinetochore/centromere proteins. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4546. doi:10.1158/1538-7445.AM2014-4546
Abstract 4558: Resveratrol alters the kinase activity of PKG-Iα and Src family in A2780cp cells (ovarian cancer cell line with mutated p53) resulting in growth inhibition
Authors: Gorjala Priyatham , Wong Janica C. , Constantino Benjamin F.b , Johlfs Mary G. , Coffman Renee , Rosenberg Harry , Fiscus Ronald
Published: May 2015

Epithelial ovarian cancer is a leading cause of death in gynecological cancer patients. One major hurdle in treating ovarian cancer is drug resistance to commonly-used chemotherapy like cisplatin. Resveratrol (3,5,4′-trihydroxy-trans-stilbene), a natural polyphenol found in foods/drinks like grapes, berries, peanuts and red wine, can inhibit tumor growth. Our lab has previously shown that resveratrol down-regulates expression of protein kinase G type-I (PKG-I) in cancer cells, which correlated with decreased cell proliferation and increased apoptosis. For the present study, we used A2780cp cells, which are cisplatin-resistant epithelial ovarian cancer cells with mutated p53. Our data show that resveratrol did not induce apoptosis even at a higher concentration of 500 µM. However, resveratrol caused concentration-dependent decreases (down to 50% of control levels at 100 µM) of both DNA synthesis and cell adhesion. We also observed a concentration-dependent increase in protein expression levels of PKG-I to 3 times of control levels at 50 µM resveratrol, but a drop at 100 µM resveratrol. Though total Src remained fairly unchanged, pSrcY416 (phosphorylation at tyrosine-416) and pSrcS17 (phosphorylation at serine-17), indicators of Src activation, decreased at 50 and 100 µM resveratrol [assessed by ultrasensitive quantitative NanoPro 1000 (ProteinSimple), a new state-of-the-art capillary electrophoresis-based protein analysis system]. Decreased pSrcY416 levels (44% of control levels at 100 µM resveratrol) suggest that the kinase activity of Src family kinases are decreased at the higher concentrations of resveratrol that cause inhibition of ovarian cancer cell proliferation. These results suggest that resveratrol can be effective in decreasing cell proliferation in chemoresistant ovarian cancer cells and that the mechanism of action of resveratrol may involve a down-regulation of the protein expression levels of PKG-I and the typrosine-kinase activity of Src family of kinases.

Citation Format: Priyatham Gorjala, Janica C. Wong, Benjamin F.b Constantino, Mary G. Johlfs, Renee Coffman, Harry Rosenberg, Ronald Fiscus. Resveratrol alters the kinase activity of PKG-Iα and Src family in A2780cp cells (ovarian cancer cell line with mutated p53) resulting in growth inhibition. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4558. doi:10.1158/1538-7445.AM2014-4558
Abstract 4561: The antihistamine cyproheptadine induces cell apoptosis through inhibition of β-catenin signaling pathways in urothelial carcinoma
Authors: Hsieh Hsiao-Yen , Wang Yuan-Hung , Shen Cheng-Huang , Chen Shiu-Yi , Chan Michael W. , Hsu Cheng-Da
Published: May 2015

Recent studies have revealed that the cyproheptadine is a novel therapeutic agent for the treatment of malignancy, such as myeloma, leukemia and HCC. However, its effect on urothelial carcinoma cell is still unknown. The aim of this study was to investigate the effect and underline mechanism of cyproheptadine on UC. We used cell viability assay to investigate the drug effect on two UC cell lines, TSGH8301 and BFTC905, and found the IC50 which was about 55 μM. After treatment of cyproheptadine, cell cycle and apoptosis status of the UC cells were determined by flow cytometry and indicated cell cycle arrest in G0/G1 phase and induction of apoptosis. We further investigated the mechanism of the drug effect using western blot analysis and found that cyproheptadine induced downregulation of cyclin-D1 and c-Myc expression through the inhibition of β-catenin signaling pathway in UC cells. More importantly, Inhibition of β-catenin signaling pathway was related to downregulation of CCRK protein in TSGH8301 cell line, but through activation of IDAX protein leading to inhibition of β-catenin signaling pathway in BFTC905 cell line. These novel results demonstrated that cyproheptadine suppressed the β-catenin signaling pathway and induced UC cell apoptosis.

Citation Format: Hsiao-Yen Hsieh, Yuan-Hung Wang, Cheng-Huang Shen, Shiu-Yi Chen, Michael Wy Chan, Cheng-Da Hsu. The antihistamine cyproheptadine induces cell apoptosis through inhibition of β-catenin signaling pathways in urothelial carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4561. doi:10.1158/1538-7445.AM2014-4561
Abstract 4563: Mechanism of action of Ibrutinib in mantle cell lymphoma
Authors: Sun Zhijian , Zhou Dongping , Wei Min , Luo Lusong
Published: May 2015

Mantle cell lymphoma (MCL), accounting for about 5-6% of non-Hodgkin lymphoma (NHL), is one of the lymphomas with very poor prognosis (median survival 3-5years).Ibrutinib, a newly approved Bruton's tyrosine kinase (BTK) inhibitor, showed remarkable clinical benefits in MCL. However, the mechanism underlining the clinical activity is not well understood. In this study, we identified MCL cell lines that were sensitive and resistant to ibrutinib. We showed that ibrutinib induced G1 cell cycle arrest in sensitive cell lines at 3-day treatment and eventually led to cell death after prolonged compound incubation. Physiologically relevant low concentration of ibrutinib inhibited phosphorylation of IKB(S32), p65/RelA(S536) and nuclear translocation of NF-κB components. Inhibition of NF-κB signaling decreased IRF4 and c-Myc level, which was accompanied with decrease of CDK6 and phosphor-RB (S807/811) level, meanwhile elevated p27/Kip1. Activation of NF-κB pathway with PKC agonist rescued ibrutinib induced G1 cell cycle arrest and eventually attenuated the cell killing effect. IRF4 phospho-RB and CDK6 level were also rescued by PKC agonist. BTK/PKC/NF-κB pathway and PI3K/AKT/mTOR pathway showed compensatory effect in MCL cell lines. Activating NF-κB pathway with PKC agonist compensated the cell killing effects of idelalisib or everolimus. Additionally, we showed that ibrutinib synergized with idelalisib or everolimus on cell killing of MCL cell lines, suggesting combination therapeutic strategies for MCL.

Citation Format: Zhijian Sun, Dongping Zhou, Min Wei, Lusong Luo. Mechanism of action of Ibrutinib in mantle cell lymphoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4563. doi:10.1158/1538-7445.AM2014-4563
Abstract 4575: Structure-based design, synthesis and biological testing of highly potent semi-synthetic epipodophyllotoxin-derived hybrid etoposide analogs
Authors: Hasinoff Brian B.
Published: May 2015

Drugs that target DNA topoisomerase II and interact with DNA represent two mechanistically distinct and clinically important classes of anticancer drugs. Using a structure-based design approach a series of epipodophyllotoxin-derived hybrid etoposide analogs were designed, synthesized and biologically characterized. It was hypothesized that combining an epipodophyllotoxin moiety that could interact with specific protein residues on topoisomerase II and DNA-interacting moieties in the same molecule would produce much stronger binding and yield more potent and wide spectrum cytotoxic anticancer drugs. Because the hybrids have unique dual targeting mechanisms, cancer cells should be much less able to develop resistance. The most potent hybrid had a mean GI50 in the NCI-60 cell screen much lower than etoposide. The compounds were much less cross-resistant to a K562-derived etoposide-resistant cell line. All of the hybrids tested targeted topoisomerase II as evidenced by their ability to inhibit kDNA decatenation, to produce linear DNA in a DNA cleavage assay and to form covalent topoisomerase II-DNA covalent complexes in a cellular ICE assay. The DNA double strand breaks the hybrids produced in a cellular γH2AX assay and the G2/M cell cycle blocks were also characteristic of topoisomerase II-targeted drugs. Cell cycle analysis was also used to characterize the targets. A COMPARE analysis indicated that the hybrids had NCI 60-cell growth inhibition profiles of both etoposide and the DNA-interacting moieties from which they were derived. This data supports the conclusion that the hybrids displayed characteristics that were consistent with having targeted both topoisomerase II and DNA. Support: CIHR and a Canada Research Chair in Drug Development.

Citation Format: Brian B. Hasinoff. Structure-based design, synthesis and biological testing of highly potent semi-synthetic epipodophyllotoxin-derived hybrid etoposide analogs. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4575. doi:10.1158/1538-7445.AM2014-4575
Abstract 4597: Characterization and targeting of radiation-inducible neoantigens in multiple cancer types
Authors: Muhoro Lincoln , Yan Heping , Hunn Jeremy , Thotala Dinesh , Ferraro Daniel , Hallahan Dennis
Published: May 2015

Targeted therapy remains one of the biggest challenges in the effective treatment of cancer. Due to their heterogeneity, not all tumors respond to a particular treatment regimen with similar efficacy. Thus, there is an urgent need to develop targeted therapies that are not only highly specific to cancer subtypes but also have minimal toxicity towards normal tissue. Currently, development of anti-cancer antibodies is limited to antigens that are either overexpressed or antigens that are only expressed in a few patients. This limits both the number of available targets and their selectivity for tumors. Using phage display technology, we have identified proteins that are induced by clinical doses of ionizing radiation (IR): tax-interacting protein 1 (TIP-1) and TATA box-associated factor 15 (TAF-15). Following IR treatment, cell surface expression of both TIP-1 and TAF-15 is enhanced in a number of human cancers, including brain, lung, breast and pancreatic cancer. The underlying premise is that IR induces a stress response resulting from DNA strand breaks and activation of ATM repair pathways. After IR stress, these antigens translocate to the surface where they can be targeted for therapeutic purposes. These radiation-inducible neoantigens present a new synergistic model for the treatment of cancer using IR. In this study, we characterized both radiation-inducible targets in vitro and in vivo. Western blotting, immunohistochemistry and flow cytometry data all demonstrated increased cell surface expression of both TIP-1 and TAF-15 after irradiation. We also developed a monoclonal antibody and a single chain antibody against TIP-1 and are currently in the process of developing antibodies against TAF-15. ELISA and near infra-red whole mouse imaging results demonstrate high affinity and high specificity of our lead anti-TIP-1 antibody and anti-TAF15 peptide in lung, brain, breast and pancreatic cancer. Dosimetry studies using immunofluorescence and Western blot analysis show a dose-dependent increase in expression of TIP-1 and TAF15, suggesting that these proteins may play a key role in the cancer cell's response to radiation stress. We are currently in the process of characterizing the mechanisms of how these proteins contribute to radioresistance and cancer survival processes such as proliferation, apoptosis and metastasis.

Citation Format: Lincoln Muhoro, Heping Yan, Jeremy Hunn, Dinesh Thotala, Daniel Ferraro, Dennis Hallahan. Characterization and targeting of radiation-inducible neoantigens in multiple cancer types. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4597. doi:10.1158/1538-7445.AM2014-4597
Abstract 4598: GP130 as a novel therapeutic target in il-6-dependent cancers
Authors: Xiao Hui , Bian Yang , Zhao Chengguang , Lin Li , Jou David , Li Huameng , Li Chenglong , Lin Jiayuh
Published: May 2015

The IL-6/GP130 signaling pathway is critical for the survival and progression of cancer cells of multiple cancer types. We report here the new discovery of Raloxifene (Marketed as Evista by Eli Lilly and Company) and Bazedoxifene [Marketed as Duavee (conjugated estrogens / bazedoxifene) by Pfizer] as novel inhibitors of IL-6/GP130 protein-protein interactions using multiple ligand simultaneous docking and drug repositioning approaches. Multiple drug scaffolds were simultaneously docked into binding hot spots of GP130 D1 domain by multiple ligand simultaneous docking to compete with the key interacting residues of IL-6, followed by tethering to generate virtual hit compounds. Similarity searches of virtual hits on drug databases identified Raloxifene and Bazedoxifene as potential inhibitors of IL-6/GP130 interaction. Both Raloxifene and Bazedoxifene are known as selective estrogen receptor modulators and we identified their novel function as GP130 inhibitors. Raloxifene and Bazedoxifene inhibited IL-6 mediated induction of STAT3 phosphorylation in cancer cells, which confirm their ability to inhibit IL-6/GP130 signaling. In contrast, Raloxifene and Bazedoxifene did not inhibit the induction of STAT1 phosphorylation by IFN-γ in cancer cells indicating their selectivity. Raloxifene and Bazedoxifene also inhibited STAT3 phosphorylation and resulted in apoptosis in cancer cells expressing elevated IL-6 levels. In addition, addition of excess IL-6 reversed the expression level of STAT3 phosphorylation which was decreased by Raloxifene or Bazedoxifene supporting that both FDA-approved drugs act through IL-6/GP130 signaling. Compared with an original GP130 inhibitor MDL-A whose IC50 values were 108-216 µM in cancer cell lines, Raloxifene and Bazedoxifene were found more potent in inhibiting cell viability which the IC50 values for Raloxifene and Bazedoxifene were 6.9-8.9 µM and 3.4-4.4 µM respectively in the same cancer cell lines expressing elevated IL-6 levels. Furthermore, both Raloxifene and Bazedoxifene significantly suppressed the tumor growth of human RH30 sarcoma cells in mouse xenograft model in vivo. These findings suggest that FDA-approved drugs Raloxifene and Bazedoxifene may serve as novel GP130-targeting therapeutic drugs in IL-6 dependent cancers.

Citation Format: Hui Xiao, Yang Bian, Chengguang Zhao, Li Lin, David Jou, Huameng Li, Chenglong Li, Jiayuh Lin. GP130 as a novel therapeutic target in il-6-dependent cancers. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4598. doi:10.1158/1538-7445.AM2014-4598
Abstract 4607: Stathmin is involved in the maternal embryonic leucine zipper kinase pathway and impacts in the outcome of glioblastoma
Authors: Uno Miyuki , Oba-Shinjo Sueli Mieko , Silva Roseli , Gimenez Marcela , Rosa Jose Cesar , Marie Suely Kazue Nagahashi
Published: May 2015

INTRODUCTION: Maternal Embryonic Leucine Zipper Kinase (MELK) is a serine/threonine kinase involved in several cell processes, including the cell cycle, differentiation, proliferation, and apoptosis. MELK has also shown multiple features consistent with the potential utility as an anticancer target, including in astrocytomas. However, the detailed downstream signaling pathway of MELK in cancer cells is still not fully understood and the putative role of MELK in astrocytomas remains unclear. The discovery of proteins involved in the MELK pathway might be important to develop additional small molecular compound to improve effectiveness. PURPOSE: This study aim to analyze proteins and/or genes involved in the MELK pathway in the tumorigenesis of gliomas. MATERIAL AND METHODS: Analysis of the expression profile of a panel of protein relevant to the process of tumorigenesis that MELK is involved was performed by Proteomic analysis by two dimensional electrophoresis and mass spectrometry for human glioma cell line U87MG transfected with siRNA for the MELK compared to U87MG transfected with non-target control (NTC) cells. Analysis and validation of gene expression was performed by Quantitative real time PCR (qRT-PCR) using SYBR Green method in a series of 153 astrocytomas of different malignant grade (23 AGI, 26 AGII, 18 AGIII and 86 AGIV or glioblastoma - GBM) and 22 non neoplastic (NN) brain samples. RESULTS: We identified Stathmin/oncoprotein 18 (STMN1) involved in the cell cycle, as one of twelve proteins differentially expressed, with lower expression when MELK was silenced. A significant higher expression of STMN1 in malignant diffusely infiltrative astrocytomas when compared to non-invasive pilocytic astrocytoma was found (p<0.0001). Strong correlation for MELK and STMN1 associated expression was observed (r=0.741, p<0.0001) in GBM. STMN1 hyperexpression alone impacted at GBM outcome with an overall survival of 5.4 months in contrast to 13.4 months when both genes were hypoexpressed (log rank=0.002). CONCLUSIONS: Thus, our results showed that STMN1 is downstream in the MELK pathway and might play an important role in astrocytoma tumorigenesis.

Citation Format: Miyuki Uno, Sueli Mieko Oba-Shinjo, Roseli Silva, Marcela Gimenez, Jose Cesar Rosa, Suely Kazue Nagahashi Marie. Stathmin is involved in the maternal embryonic leucine zipper kinase pathway and impacts in the outcome of glioblastoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4607. doi:10.1158/1538-7445.AM2014-4607
Abstract 461: Modulation of estrogen receptor (ER) and androgen receptor (AR) by a modified CRISPR-Cas9 system
Authors: Zhou Nanjiang , Mo Yin-Yuan
Published: May 2015

Clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated (Cas) system is a bacterial adaptive immune defense against foreign nucleic acids from viruses, plasmids, and other mobile genetic elements. There are three types of CRISPR systems; type II uses single effector enzyme, Cas9, to cleave double-stranded DNA (dsDNA). Because of this unique feature, CRISPR/Cas9 system has been widely used as a genetic engineering tool from bacteria to mammals. In this system, Cas9 nuclease can bind and cleave genome directed by sequence-specific guide RNA (gRNA). While the wild type Cas9 can cleave double stranded DNA, the mutant Cas9 at D10A and H804A can only bind to but cannot cleave the DNA. Given this unique feature, we tried to repurpose Cas9 as a gene activation or suppression tool. To achieve this, we generated DMCas9 (double-mutant Cas9), and fused it with VP16AD (VP16 activation domain) or VP64AD (VP64 activation domain) to explore its utility for gene activation. To generate a Cas9-based gene silencing tool, we fused DMCas9 with Krab, a well-known protein domain with suppression function. Using a reporter system carrying a specific sequence that was recognized by a given gRNA and mCherry as a readout, we demonstrated that co-expression of VP16AD-DMCas9 and specific gRNAs leads to a at least three-fold induction of mCherry signal; in contrast, DMhCas9-Krab along with the specific gRNA resulted in a significant suppression of mCherry activity. Furthermore, we showed that DMCas9-Krab combined with gRNAs suppressed endogenous expression of AR and ER in MCF-7 cells, causing cell growth inhibition. Together, these results demonstrate the feasibility of Cas9-based gene activation and gene suppression.

Citation Format: Nanjiang Zhou, Yin-Yuan Mo. Modulation of estrogen receptor (ER) and androgen receptor (AR) by a modified CRISPR-Cas9 system. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 461. doi:10.1158/1538-7445.AM2014-461
Abstract 4614: Identification of two salinomycin binding targets in neuroblastoma
Authors: Zhou Shuang , Wang Fengfei , Xiang Shi-Hua , Wong Eric T. , Muhonen Wallace W. , Fonkem Ekokobe , Hsieh Tze-chen , Li David W. , Zhang Ruiwen , Shabb John B. , Wu Joseph M. , Wu Min , Wu Erxi
Published: May 2015

Salinomycin, a widely used anti-coccidial agent isolated from a Streptomyces albus strain, was recently identified from a library of 16,000 natural and commercial chemical compounds based on its highly selective inhibitory effect on breast cancer stem cells (CSCs), with more than 100-fold greater potency than paclitaxel. Salinomycin also exhibits cytotoxic effects on other types of cancer cells and CSCs and overcomes drug resistance. However, the exact mechanism of salinomycin, especially its direct binding target(s), and its effects on neuroblastoma (NB) are yet not known.

NB is one of the most common solid tumors in children and infants, and a leading cause of mortality in children. Despite advances in diagnosis and treatment, currently, 35% of patients with NB remain incurable. In addition, the majority survivors of NB suffer from long-term side effects of current therapies and are at risk for disease relapse or getting a second, different cancer. More effective therapies are pressingly needed. Since the existence of CSCs in human NB cell lines and NB tumors from patients has been well documented, and has been closely associated with chemoresistance or tumor relapse, therapeutic targeting of NB CSCs may be a critical novel approach to conquer the challenges in NB therapy.

Aiming to improve NB therapy, we examined the efficacy and mechanism of salinomycin in human NB cells. Our study showed that salinomycin markedly inhibits NB cell proliferation and tumorsphere formation. Treatment of salinomycin induced G2 cell cycle arrest with an up-regulation of Cyclin A and a down-regulation of p21 protein levels. We further identified TIF1β and NCL as novel direct binding proteins of salinomycin by using comprehensive methods, including chemical proteomics and functional genomics. Furthermore, by analyzing tumor samples from a cohort of 88 NB patients, we found that the elevated level of these proteins is associated with poor prognosis for NB patients. Thus, our results indicate salinomycin is a potential new therapeutic agent and TIF1β and NCL as new therapeutic targets/biomakers for NB therapy.

Citation Format: Shuang Zhou, Fengfei Wang, Shi-Hua Xiang, Eric T. Wong, Wallace W. Muhonen, Ekokobe Fonkem, Tze-chen Hsieh, David W. Li, Ruiwen Zhang, John B. Shabb, Joseph M. Wu, Min Wu, Erxi Wu. Identification of two salinomycin binding targets in neuroblastoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4614. doi:10.1158/1538-7445.AM2014-4614
Abstract 4618: Therapeutic targeting for sphingosine kinase 1 in epithelial ovarian cancer
Authors: Lee Jeong-Won , Ryu Ji Yoon , Jeon Hye-Kyung , Park Young-Ae , Cho Young-Jae , Choi Jung-Joo , Lee Yoo-Young , Kim Tae-Joong , Choi Chel Hun , Kim Byoung-Gie , Bae Duk-Soo
Published: May 2015

Purpose: Sphingosine kinase 1 (SK1) is over-expressed in various human cancers with pro-growth effects and its inhibitors has been suggested potential anti-cancer agents. This study was designed to investigate the therapeutic potential of SK1 inhibitor in epithelial ovarian carcinoma (EOC).

Experimental design: The expression of SK1 protein was evaluated using immunohistochemistry in patients with EOC. EOC cell lines (A2780, SKOV3ip1, A2780-CP20, SKOV3-TR, ES2 and RMG2) were used in this study to test SK1 siRNA or inhibitors. We used two kinds of SK inhibitor including SK inhibitor (for both SK1 and 2) and FTY720 (specifically inhibiting SK1) to check cell proliferation, apoptosis, angiogenesis and invasion using MTT, FACS, ELISA and wound-healing assay, respectively. Furthermore, in vivo experiments were performed to test the FTY720 on tumor growth in orthotopic EOC mouse model.

Results: The expression of SK1 protein in primary EOC tissues was strongly observed in all of patients; however, there was no expression of SK1 protein in the normal ovarian epithelium (n=5). Blocking SK1 by its siRNA or inhibitors significantly affected cell proliferation, apoptosis, angiogenesis and invasion in A2780-PAR and

SKOV3ip1 cells. SK1 inhibitors led to decrease of SK enzymatic activity in cells. Furthermore, utilizing the in vivo animal model (A2780-PAR), FTY720 treatment significantly decreased the total tumor weight compared with control (P < 0.05).

Conclusions: These results show that therapeutic targeting of SK1 with its inhibitor could have potentials of novel therapeutics for EOC.

Citation Format: Jeong-Won Lee, Ji Yoon Ryu, Hye-Kyung Jeon, Young-Ae Park, Young-Jae Cho, Jung-Joo Choi, Yoo-Young Lee, Tae-Joong Kim, Chel Hun Choi, Byoung-Gie Kim, Duk-Soo Bae. Therapeutic targeting for sphingosine kinase 1 in epithelial ovarian cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4618. doi:10.1158/1538-7445.AM2014-4618
Abstract 462: Chromatin signatures of DLBCL subtypes
Authors: McCord Ron , Field Megan , Jordan Philip , Hunter Ewan , Akoulitchev Alexandre , Mundt Kirsten E.
Published: May 2015

Diffuse Large B-cell Lymphoma (DLBCL) is a common and aggressive type of Non-Hodgkin's Lymphoma. Gene expression profiling long ago identified two distinct subtypes: Activated B-Cell (ABC) and Germinal Center B-Cell (GCB), reflecting the biology associated with the cell of origin of the lymphoma (Alizadeh et al, Nature 2000).

These subtypes are histologically non distinguishable, yet are associated with distinct outcomes. Emerging clinical data also suggests that they are predictive of outcome for novel therapeutics, including a BTK inhibitor, a proteasome inhibitor and an immune modulatory agent. Reliable diagnosis of the DLBLC subtype may therefore become a requirement for choosing the best treatment for DLBCL patients.

Since gene regulation is known to be associated with changes in chromatin structures, we decided to investigate the chromatin conformation around key regulators of ABC/ GCB biology using a Chromatin Conformation Capture Assay (3C). For this assay, chromatin loops are cross-linked; the DNA is digested and re-ligated, joining topologically proximal ends that may be very distant along the length of the chromosome into distinct novel fusion products. After reversing the cross-links, the presence of these novel DNA fusions can be detected by PCR, resulting in a binary read-out. Composites of these PCR products form a signature that may be characteristic of disease, disease state or subtype. The EpiSwitchTM technology has been optimized to identify these epigenetic patterns not only in tissue, but also from blood borne tumor DNA.

To investigate characteristic changes in chromatin conformation in ABC- and GCB- DLBCL cell lines, we focused on key genes known to be differentially expressed in the two sub-types, including Bcl-6, IRF-4 and LMO-2 (Wright et al. PNAS 2003,). We mapped several interactions indicating that these loci are subject to epigenetic regulation. We also applied the assay to blood from DLBCL patients and from healthy volunteers to exclude background signals from normal B-cell maturation. The assay was able to detect the same signatures as seen in cell lines.

These results demonstrate the potential of this technology to detect DLBCL subtype specific chromatin conformation associated biomarkers in patient blood.

Citation Format: Ron McCord, Megan Field, Philip Jordan, Ewan Hunter, Alexandre Akoulitchev, Kirsten E. Mundt. Chromatin signatures of DLBCL subtypes. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 462. doi:10.1158/1538-7445.AM2014-462
Abstract 464: Methylase meets phosphatase: roles of PRMT and EYA1 in neuroblastoma
Authors: Li Xingguo , Wang Simeng , Schor Nina F.
Published: May 2015

High-risk neuroblastoma is a lethal childhood tumor characterized by increased oncoprotein MYCN expression. As strategies for modulation of MYCN function itself have proved challenging, targeting of MYCN's downstream effectors may provide an alternative option. Protein arginine methyltransferase 1 (PRMT1) has been reported as a MYCN target gene in neuroblastoma; high expression of PRMT1 is significantly associated with a poor prognosis. Importantly, PRMT1-deficient cells have been shown to be hypersensitive to chemotherapy-induced DNA damage, suggesting that targeting PRMT1 may overcome drug resistance for patients with advanced neuroblastoma. However, the underlying mechanisms of how PRMT1 contributes to chemoresistance and tumor progression are still largely unknown. We report here that PRMT1 forms a novel complex with EYA1, a phosphatase and transcriptional coactivator that may serve as the crucial determinant of survival versus apoptotic responses to DNA damage through dephosphorylation of histones. Interestingly, we found that EYA1 is highly expressed in advanced neuroblastoma and that PRMT1 physically associates with EYA1 through direct interaction. Further, rendering EYA1 refractory to methylation by PRMT1 abolishes its phosphatase activity. These findings suggest that PRMT1 methylase, EYA1 phosphatase, and PRMT1-EYA1 may be druggable targets for the therapy of neuroblastoma.

Citation Format: Xingguo Li, Simeng Wang, Nina F. Schor. Methylase meets phosphatase: roles of PRMT and EYA1 in neuroblastoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 464. doi:10.1158/1538-7445.AM2014-464
Abstract 465: EVI1 transcriptionally represses SLUG to regulate epithelial-mesenchymal transition (EMT) in human colon cancer
Authors: Nayak Kasturi B. , Chakraborty Soumen
Published: May 2015

Ecotropic viral integration site-1 (EVI1) is an oncogenic transcription factor that is frequently implicated in hematological malignancies and in some solid tumors. Deregulated expression of EVI1 impairs several pathways including proliferation, differentiation, apoptosis and cell cycle through different mechanisms in different cell types. EVI1 was found to be overexpressed in 53% of human colorectal cancer samples, 100% of colon adenocarcinoma samples, 100% of human colon cancer cell lines and its presence might affect disease progression and sensitivity to chemotherapy. To investigate the stage specific expression pattern of EVI1 in colon cancer patient samples, we analyzed two of the publicly available microarray datasets (GEO database) of colon cancer patients and observed that although EVI1 is upregulated in the initial stage of the disease, the expression level decreases with the progression of the disease. In cancer cells loss of epithelial adhesion molecule E-cadherin is considered to be a fundamental event in EMT. To decipher whether EVI1 is involved in EMT, we checked five transcription factors (SLUG, SNAIL, TWIST, ZEB1 and ZEB2) which controls the expression of E-cadherin in cancer cells. Significant negative correlation was observed between the expression levels of EVI1 and all the above transcription factors in colon cancer patient samples. Recently it was shown by using ChIP sequencing that EVI1 target SLUG and SNAIL in SKOV3 cells. To understand whether EVI1 directly binds to both SLUG and SNAIL, we did an in silico analysis and searched for EVI1 binding site(s) in the functional promoter region of both SLUG and SNAIL. Although we did not observe any binding site in SNAIL promoter region; we observed two EVI1 binding sites in the SLUG promoter region. To validate the predicted target sites in vivo, we did a ChIP assay and found that EVI1 binds to one of the site and not both of them. To determine the transcriptional regulation, we cloned the promoter region of SLUG (-1100 bp) in pGL3 vector and did luciferase assay, which revealed that EVI1 significantly represses SLUG activity in EVI1 transfected cells. Analysis of gene expression databases corroborates the above finding and shows that EVI1 negatively regulates SLUG expression in each stage of the disease as well as in colon cancer cell lines. We have worked on the functional aspects of the binding in terms of EMT which will be presented in the poster.

Citation Format: Kasturi B. Nayak, Soumen Chakraborty. EVI1 transcriptionally represses SLUG to regulate epithelial-mesenchymal transition (EMT) in human colon cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 465. doi:10.1158/1538-7445.AM2014-465
Abstract 4652: Cerebrospinal fluid pharmacokinetics and pharmacodynamics following high-dose erlotinib treatment in brain cancer patients
Authors: Pastorino Sandra , Pingle Sandeep C. , Langley Emma , Kim Phillip , Juarez Tiffany , Jiang Pengfei , Tucker Christopher , Yang Txheng , Saria Marlon , Singh Sharat , Kesari Santosh
Published: May 2015

Patients with non-small cell lung cancer (NSCLC) who develop leptomeningeal metastases show significant clinical improvement following high-dose gefitinib therapy. Based on this data, we aimed to explore the effect of high-dose erlotinib on clinical response and CNS penetration in primary or metastatic CNS cancer.

We selected patients who had received prior therapy for either primary glioma or for NSCLC with metastases to the brain and treated them with an alternate dosing regimen of erlotinib. Blood and cerebrospinal fluid (CSF) samples were collected at various time points to assess levels of erlotinib. We found that CSF concentrations of erlotinib were generally 2% that of plasma concentrations. Among the five patients examined, CSF concentrations of erlotinib reached up to 131 nM when high doses were administered at 600 to 1200 mg every four to six days. Additionally, drug clearance was decreased with an increased dosing regimen in one of the patients. In one patient, the area under the curve (AUC) estimations showed a 24% increase in the CSF versus only a 2% increase in the plasma following an increase from 150 mg to 600 mg of erlotinib. Moreover, longitudinal CSF samples were evaluated for expression and activation of EGFR as well as various other receptor tyrosine kinases, such as ErbB2, ErbB3, cMET and IGF1R, using the highly sensitive multiplexed immunoassay CEER© platform. The latter provides insight into the molecular makeup of the CNS cancer throughout the therapeutic regimen.

Our clinical data on patients receiving erlotinib therapy for primary and metastatic CNS disease suggests that increased dosing of erlotinib, administered as a pulse dose every four to six days leads to increased CSF drug concentrations. In spite of the variations seen among different patients, we conclude that higher erlotinib dosing regimens can provide enhanced CNS penetration that may prove to be more effective in primary and metastatic CNS cancers especially when coupled with real-time molecular monitoring of the disease to help guide the clinician during the course of treatment.

Citation Format: Sandra Pastorino, Sandeep C. Pingle, Emma Langley, Phillip Kim, Tiffany Juarez, Pengfei Jiang, Christopher Tucker, Txheng Yang, Marlon Saria, Sharat Singh, Santosh Kesari. Cerebrospinal fluid pharmacokinetics and pharmacodynamics following high-dose erlotinib treatment in brain cancer patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4652. doi:10.1158/1538-7445.AM2014-4652
Abstract 4654: Plasma, tissue and tumor pharmacokinetics of PM060184 in NSCL xenograft mouse model
Authors: Pernice Tiziana , Bishop Alan , Cataluña Oscar , Palomares Mandy , Lopez Raquel , Núñez Práxedes , Cuevas Carmen , Guillén Maria José , Aviles Pablo M.
Published: May 2015

Background: PM060184 is new marine derived tubulin-binding agent originally isolated from the sponge Lithoplocamia lithistoides. PM060184 has shown potent antitumor activity in vivo against a panel of different tumor xenografted models such as colon, gastric, NSCLC, prostate and renal. Also, PM060184 has demonstrated to have a potent in vitro and in vivo activity in P-gp overexpressing cells. PM060184 is currently under evaluation in Phase I clinical studies in patients with advanced cancer diseases.

Material and Methods: Athymic female nu/nu mice were subcutaneously implanted with H460 (NSCLC). Tumor bearing animals were randomly allocated into experimental groups (efficacy or pharmacokinetics -PK-). The animals belonging to the efficacy study received three iv doses in a weekly (q7dx3) schedule of PM060184 at 16 mg/kg or placebo. The antitumor activity was followed by the change in tumor volume for treated and placebo groups. The PK group received a single dose of PM060184 at 16 mg/kg. Then, plasma, tissue (brain, spleen, lung, muscle and heart) and tumor samples (N=3-4/sampling time) were collected at different times and up to 96 h post-administration. Tissue and tumor samples were diluted and homogenized using Precellys®24 bead beating technology. Once homogenized the samples were processed as per plasma. PM060184 was detected in plasma, tissue and tumor extracts by electrospray ionization/tandem mass spectrometry after extraction by supported liquid extraction (SLE).

Results: The mean maximum plasma concentration (Cmax) was 2,452.5 ng/mL. The area under curve (AUC) was 2,963 ng*hr/mL. The plasma clearance was 5,398.5 mL/hr/kg. The volume of distribution at steady state was 880.7 mL/kg. The terminal half-life was 4.7 hr. The AUC0-tlast values for brain, spleen, lung, muscle and heart were 216.2, 7,402.9, 3,857.7, 2,614.6 and 2,929.5 ng*hr/g, respectively; however the AUC value for H460 xenografted tumors was 51,321.4 ng*hr/g (tlast is 96 hours for tumor, 8 hours for brain and 24 hours for the other tissues).

Conclusion: PM060184 showed similar plasma and tissue pharmacokinetics properties as well as preferential tumor distribution until 96 h after an iv administration in mice bearing H460 xenografted tumors.

Citation Format: Tiziana Pernice, Alan Bishop, Oscar Cataluña, Mandy Palomares, Raquel Lopez, Práxedes Núñez, Carmen Cuevas, Maria José Guillén, Pablo M. Aviles. Plasma, tissue and tumor pharmacokinetics of PM060184 in NSCL xenograft mouse model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4654. doi:10.1158/1538-7445.AM2014-4654
Abstract 4664: Single-cell transcriptome analysis on lung squamous cell carcinoma derived from a patient
Authors: Kim Kyu-Tae , Lee Hye-Won , Joo Kyeung Min , Kim Sang-Chul , Lee Jin-Ku , Chang Nakho , Sa Jason K. , Cho Yu Jin , Nam Do-Hyun , Lee Hae-Ock , Park Woong-Yang
Published: May 2015

Distinct genomic signature of a patient's cancer could provide a clue to design and predict accurate therapeutic responses to targeted medicine. Current approaches regarding heterogeneous cancer cells en masse as a pooled population, however, hardly reflect complete genomic landscape of tumor diversity, missing potentially important minor clones' implications regarding metastasis and drug resistance. Here we report a comprehensive analysis to discover different patterns of subclonal behaviors in functional modalities and signaling pathways out of heterogeneous population derived from a patient with lung squamous cell carcinoma, using single-cell mRNA sequencing. As a result of nonnegative matrix factorization as a clustering, we identified discrete 4 subgroups with a major group in accordance with overall expression profile of a pooled population of cells, and the other 3 minor groups that were masked in a pooled sample. Genomic variants were also reliably identified and compared across single cells. We integrated these genomic signatures from single cells to address the different responses in drug screening. Together, our approach provides insights into the more accurate strategy to identify minor but potentially important subclones that are relevant to drug resistance.

Citation Format: Kyu-Tae Kim, Hye-Won Lee, Kyeung Min Joo, Sang-Chul Kim, Jin-Ku Lee, Nakho Chang, Jason K. Sa, Yu Jin Cho, Do-Hyun Nam, Hae-Ock Lee, Woong-Yang Park. Single-cell transcriptome analysis on lung squamous cell carcinoma derived from a patient. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4664. doi:10.1158/1538-7445.AM2014-4664