bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4288: RASSF1C, unlike RASSF1A, reduces TNF-α induced phosphorylation of MST1/2.
Authors: Amaar Yousef G. , Firek Matthew , Reeves Mark E.
Published: August 2014

Introduction: Recently, RASSF1A has been shown to mediate the apoptotic effects of TNF-α by interacting with the mammalian sterile 20-like kinase 1 and 2 (MST1/2) through the Salvador/Rassf/Hippo (SARAH) domain leading to MST1/2 phosphorylation activation of apoptosis through the Hippo pathway. In contrast, we have previously shown that RASSF1C promotes cell proliferation and attenuates apoptosis in cancer cells. Since both RASSF1A and RASSF1C contain the SARAH domain located in their identical C-termini, RASSF1C should be capable of interacting with SARAH domain-containing proteins, and could potentially attenuate MST1/2-mediated apoptosis through the Hippo pathway. Thus, in this study we have investigated the impact of RASSF1C on MST1/2 activation/phosphorylation in presence of TNF-α, including whether RASSF1C modulates MST1/2 pro-apoptotic effects through the Hippo pathway differently from RASSF1A.

Method: Breast and lung cancer cells over-expressing RASSF1A and RASSF1C were used in this study. Cells were cultured in the proper media and treated with TNF-α for 12-18 hr. Cells were collected and used for Western blot analysis utilizing antibodies that detect MST and p-MST antibodies.

Results: Our preliminary data suggest that RASSF1C over-expression decreases the phosphorylation levels of MST1/2 in TNF-α-treated breast and lung cancer cells compared to those cells over-expressing RASSF1A

Conclusion: Our findings suggest that over-expression of RASSF1C in breast and lung cancer cells may attenuate the MST1/2 apoptotic effects by sequestering of MST1/2 proteins and inhibiting their activation by phosphorylation. The findings also further support our hypothesis that RASSF1C attenuates apoptosis; and RASSF1C, unlike RASSF1A, may inhibit MST1/2 activation and hence may negatively modulate the Hippo pathway.

Citation Format: Yousef G. Amaar, Matthew Firek, Mark E. Reeves. RASSF1C, unlike RASSF1A, reduces TNF-α induced phosphorylation of MST1/2. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4288. doi:10.1158/1538-7445.AM2013-4288
Abstract 4304: PPP1R1B-STARD3 fusion gene promotes gastric tumorigenesis through activation of PI3K/Akt signaling.
Authors: Yun Sun Mi
Published: August 2014

Fusion genes result from genomic aberrations such as translocation, amplification and rearrangement. They have the potential to be new targets for the diagnosis and therapeutics in several different types of cancers. To identify fusion genes in gastric cancer, we conducted RNA sequencing with 18 gastric cancer cell lines and 11 pairs of primary human gastric cancer tissues and their adjacent normal tissues. We identified PPP1R1B-STARD3 fusion gene and validated the expression of this fusion gene by Real-Time PCR in the expanded panel of the human gastric tissue samples. Interestingly, PPP1R1B-STRAD3 fusion gene was detected in approximately 30% of primary human gastric cancers, but not in adjacent matched normal gastric tissues. Overexpression of PPP1R1B-STARD3 in MKN-28 significantly increased cell proliferation and colony formation. This increased proliferation was mediated by activation of PI3K/Akt signaling. To confirm if the expression of PPP1R1B-STARD3 fusion gene affects the tumorigenic capacity of gastric cancer cells in vivo, we performed the xenograft assay with MKN-28 cells that stably express PPP1R1B-STARD3 fusion gene. Overexpression of the fusion gene enhanced the MKN-28 tumor growth in nude mice. Hence, PPP1R1B-STARD3 fusion gene overexpression gives growth advantage in vitro and in vivo. Taken together, these findings indicate that PPP1R1B-STRAD3 fusion gene increased cell proliferation through the activation of PI3K/Akt signaling in gastric cancer cells.

Citation Format: Sun Mi Yun. PPP1R1B-STARD3 fusion gene promotes gastric tumorigenesis through activation of PI3K/Akt signaling. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4304. doi:10.1158/1538-7445.AM2013-4304
Abstract 4309: Fibulin-2 suppresses tumor growth and angiogenesis through the inhibition of Erk1/2 and p65 pathways in nasopharyngeal carcinoma.
Authors: Shuen Wai Ho , Lung Maria Li
Published: August 2014

Nasopharyngeal carcinoma (NPC) has distinctive ethnic and geographic distributions, with the highest incidence in Southern China. Infection with the human herpesvirus, Epstein-Barr virus, genetic predisposition, dietary, and environmental factors are high-risk factors for the development of NPC. Evidence indicates that loss of heterozygosity on chromosome 3 is one of the early events contributing to the cancerization of normal nasopharyngeal epithelial cells. By using a chromosome 3 NotI genomic microarray, Fibulin-2 (FBLN2) mapping to chromosome 3p25.1, was previously identified as a candidate tumor suppressor. FBLN2, which is an extracellular matrix (ECM) protein, contains four domains, regions consisting of cysteine-rich and cysteine-free, anaphylatoxin-like domain, serial EGF-like repeats, and a fibulin module. FBLN2 is silenced by promoter hypermethylation in NPC. Re-expression of FBLN2 in NPC cell lines significantly reduces colony formation, cell proliferation, migration, invasion, and in vivo tumor formation and angiogenesis. In this study, the aim is to investigate the molecular mechanisms and signaling pathways of FBLN2-mediated tumor suppressive and anti-angiogenesis effects. Full-length FBLN2 was subcloned into inducible tetracycline-regulated and lentivirus systems. Phosphorylated forms of the key signaling molecules, Erk1/2 and NFκB, were found to be significantly reduced in FBLN2 stably-expressing NPC cell lines after phosphorylation target screening using Western blot analysis. FBLN2 also induces changes in the cytoskeleton arrangement, possibly through the regulation of Rho GTPases. Thus, the activities of Rho GTPases were determined by the Rho pull-down assays and immunofluorescence staining with confocal microscopy. FBLN2 is able to reduce the tumor formation ability via inhibiting Erk1/2 and NFκB pathways and to inhibit the cell migration and invasion via regulating Rho GTPase activities. FBLN2 is a secretory protein and is generally silenced in NPC, suggesting the possibility that it may be useful as a diagnostic marker and/or new target for therapeutics of NPC. Therefore, these studies provide strategic new insights into the understanding of the mechanistic role of FBLN2 in NPC and enhance our understanding of the molecular genetic basis of NPC tumorigenesis.

Citation Format: Wai Ho Shuen, Maria Li Lung. Fibulin-2 suppresses tumor growth and angiogenesis through the inhibition of Erk1/2 and p65 pathways in nasopharyngeal carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4309. doi:10.1158/1538-7445.AM2013-4309
Abstract 4320: Agtuzumab, a humanized monoclonal antibody, blocks AGR2 function through conformational epitopes around its catalytic center.
Authors: Li Dawei , Wu Zhenghua , Zhu Qi , Guo Hao , Gao Guangwei , Mashausi Dhahiri Saidi , Chen Hao
Published: August 2014

AGR2 is a member of protein disulfide-isomerase (PDI) family and plays an important role in cancer development including tumorigenesis and metastasis. Unlike the classical PDI protein with a CxxC catalytic center, AGR2 contains a CxxS center that function in Mucin2 formation on the endoplasmic reticulum. However, AGR2 has also been shown to present in blood plasma and is secreted by several cell lines. As a secreted small protein, AGR2 is particularly suitable target for antibody medicine and its functional antibody is under investigation for therapeutic potentials by several major pharmaceutical firms and academic institutions but few showed AGR2 any anti-cancer efficacy. Here we report a humanized monoclonal antibody against AGR2, Agtuzumab, with promising therapeutic potentials. This antibody blocks AGR2 function in cell migration and proliferation assays and reduced tumorigenic and angiogenic potential of xenograft tumors in nude mice. Treatment of AGR2-blocking antibody reduced the expression of VEGF and VEGFR level in xenograft tumor tissues for both human tumor and its mouse stroma cells. Scanning mutagenesis assays revealed discontinuous regions around the CxxS PDI center. on AGR2 protein that are essential for efficient binding of Agtuzumab. Our results for the first time confirmed with a potential therapeutic antibody that AGR2 can be a therapeutic target and its blocking requires the blockage of a conformational epitope. This result also explains why many monoclonal antibodies developed against peptides may not be functional against AGR2 related tumors.

Citation Format: Dawei Li, Zhenghua Wu, Qi Zhu, Hao Guo, Guangwei Gao, Dhahiri Saidi Mashausi, Hao Chen. Agtuzumab, a humanized monoclonal antibody, blocks AGR2 function through conformational epitopes around its catalytic center. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4320. doi:10.1158/1538-7445.AM2013-4320
Abstract 4326: Phosphatidylserine-targeting ‘betabodies’ for the treatment of cancer.
Authors: Huang Xianming , Ye Dan , Luster Troy , Ward E Sally , Thorpe Philip
Published: August 2014

Bavituximab is a chimeric monoclonal antibody that is being combined with chemotherapy to treat patients with lung or pancreatic cancer in randomized Phase II clinical trials. Bavituximab targets the immunosuppressive lipid, phosphatidylserine (PS), which becomes exposed on the outer membrane surface of tumor blood vessels and tumor cells in tumors responding to therapy. The antibody acts by destroying tumor vasculature and by reactivating tumor immunity. Here, we generated new PS-targeting therapeutics by fusing domains of the PS-binding plasma protein, mouse β2-glycoprotein I (β2GP1), to the Fc region of mouse IgG2a. Such ‘betabodies’ potentially have the following advantages: they bind directly to PS, and do not require a cofactor protein (β2GP1) for binding; they can be made fully human; they are smaller in size (100 KDa); and they have slower blood clearance rates. A construct was generated that bound strongly to PS-expressing cells and plates, localized to tumor vascular endothelium in vivo, and had a β-phase blood half-life of approximately five days after intravenous injection into mice as compared with one day for a murine version of bavituximab, 2aG4. Betabodies could potentially be the next generation of PS-targeting cancer therapeutics.

Citation Format: Xianming Huang, Dan Ye, Troy Luster, E Sally Ward, Philip Thorpe. Phosphatidylserine-targeting ‘betabodies’ for the treatment of cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4326. doi:10.1158/1538-7445.AM2013-4326
Abstract 4338: Tumor reversion: From bench to potential clinical applications using sertraline and thioridazine.
Authors: Amson Robert , Pece Salvatore , Di Fiore Pier Paolo , Karp Judith , Marine Jean-Christophe , Telerman Adam
Published: August 2014

This presentation aims at summarizing today's knowledge on tumor reversion (ref.1) with recent applications and new findings for potential cancer treatment. In order to study the molecular program of tumor reversion we have provided with a series of biological models. In this approach, we derived revertant cells from tumor cell lines ranging from leukemia to solid tumors such as breast, colon, lung cancer and melanoma. Using differential gene profiling, between the parental tumor cells and the revertants, over 300 genes were identified, as implicated in reversion such as Siah-1, TSAP6, PS1 and most importantly Translationally Controlled Tumor Protein (TPT1/TCTP) (ref. 2). Notably TPT1/TCTP is highly expressed in the tumor cells and almost undetectable in the revertants. Knocking down TPT1/TCTP reorganized breast cancer cells into ductal structures reminiscent of normal breast morphology. More recently, we identified a strong reciprocal repressive feedback loop between TPT1/TCTP and p53 (ref.3). P53 represses the transcription of TPT1/TCTP by binding to the promoter of TPT1/TCTP. Conversely, TPT1/TCTP interacts directly with p53, Numb and MDM2. TPT1/TCTP competes with Numb. Importantly, TPT1/TCTP stabilizes MDM2, promoting hereby MDM2-dependent ubiquitination and proteasomal degradation of p53. Screening of a large cohort of breast cancer patients (508) shows high expression of TPT1/TCTP in the most aggressive G3 tumors, predicting poor prognosis. TPT1/TCTP is also highly expressed in normal and cancer breast stem cells. Decreasing TPT1/TCTP by siRNA in a mammosphere assay significantly reduces the colony forming efficiency. Thus, TPT1/TCTP also regulates the stem cell compartment. We found two drugs, Sertraline and Thioridazine, which bind and inactivate TPT1/TCTP, restoring elevated levels of wild type p53. Pilot studies on ex vivo cells derived from AML-patients indicate that Sertraline and Ara-C act synergistically in reducing the viability of AML cells. The implication of tumor reversion in reprogramming cancer cells and in disclosing new therapeutic targets as well as its potential role in tumor heterogeneity will be discussed.

Citation Format: Robert Amson, Salvatore Pece, Pier Paolo Di Fiore, Judith Karp, Jean-Christophe Marine, Adam Telerman. Tumor reversion: From bench to potential clinical applications using sertraline and thioridazine. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4338. doi:10.1158/1538-7445.AM2013-4338
Abstract 4339: RGD-conjugated nanoparticles for targeted inhibition of adhesion and migration of integrin αvβ3-overexpressing breast cancer cells.
Authors: Shan Dan , Cai Ping , Prasad Preethy , Rauth Andrew Michael , Wu Xiao Yu
Published: August 2014

Background: Tumor cell adhesion and migration are critical in the establishment of metastasis. This study aims to develop a RGD-conjugated nanoparticle system that is able to target αvβ3 integrin receptor-overexpressing breast tumor cells while evading the liver uptake, thus enhancing treatment of cancer metastasis through inhibition of adhesion and migration of cancer cells.

Methods: The nanoparticle formulation was first optimized with a suitable concentration of RGD targeting moiety on the nanoparticle surface to maximize nanoparticle accumulation in tumor and minimize liver uptake. RGD-conjugated solid lipid nanoparticles (RGD-SLNs) were synthesized with a fatty acid lipid core and a poly(ethylene glycol) corona and decorated with varying concentrations of RGD peptides (0%, 0.5%, 1%, 5% and 10% mol/mol ratio). The quantity and kinetics of the nanoparticle binding to αvβ3 integrin receptor and in vitro cellular uptake on αvβ3 positive MDA-MB-231 cells were investigated by fluorescence microscopy and compared with the results with αvβ3 negative MCF-7 cells. The influence of RGD-SLNs on cell adhesion and migration via binding to αvβ3 integrin receptor was examined using standard adhesion and transwell migration assays. Whole animal biodistribution, tumor uptake and intratumoral distribution of quantum dot-loaded nanoparticles were investigated to identify optimized RGD-concentrations.

Results: RGD-SLNs showed specific binding for αvβ3 integrin receptors on MDA-MB-231 cells as compared to the negative control (MCF-7 cells). The RGD-SLN formed clusters at the cellular membrane and then entered the cells at a lower pace than SLN. The RGD-SLNs also exhibited higher cellular uptake compared to SLN in MDA-MB-231 cells while there was no detectable difference in uptake in αvβ3 negative MCF-7 cells. RGD concentration of 1% on the SLN surface was found to have most tumor retention and low liver uptake among all formulations. Receptor mediated uptake of RGD-SLNs reduced cell adhesion and migration towards fibronectin gradient, attributable to the occupation of the receptors by the RGD-SLN and slow recycling of the receptors to the cell surface.

Conclusions: We have optimized the RGD-SLN formulation to maximize tumor accumulation and minimize liver uptake. The RGD-SLN formulation has the potential to prevent metastasis through interference with cell adhesion and migration.

Citation Format: Dan Shan, Ping Cai, Preethy Prasad, Andrew Michael Rauth, Xiao Yu Wu. RGD-conjugated nanoparticles for targeted inhibition of adhesion and migration of integrin αvβ3-overexpressing breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4339. doi:10.1158/1538-7445.AM2013-4339
Abstract 4347: Toxicology and drug delivery by novel Cucurbit[n]uril-type molecular containers.
Authors: Hettiarachchi Gaya , Ma Da , Nguyen Duc , Isaacs Lyle , Briken Volker
Published: August 2014

Background. Many drug delivery systems are based on the ability of certain macrocyclic compounds - such as cyclodextrins - to act as molecular containers for pharmaceutical agents in water. Cucurbit[n]urils (CB[n]) are a class of molecular containers that bind to a variety of cationic and neutral species with high affinity (Ka > 10ˆ4 Mˆ-1) and therefore show great promise as a drug delivery system (Isaacs, 2009). Initial in vitro toxicity analysis demonstrated good biocompatibility of five different CB[n]-type compounds. We hypothesized that these nanocontainers can be used to increase solubility of hydrophobic small chemical compound drugs. Methodology. In this study we investigated the toxicology and bioactivity of one novel cucurbit[n]urils CB[n]-type container (named Motor1). The container induced no toxicity at concentrations of up to 10 mM in human cell lines originating from kidney, liver or blood tissue using assays for metabolic activity and cytotoxicity. Furthermore, the Motor1 container was tolerated in mice without any toxicity after intravenous dosing of up to 1.5g/kg bodyweight. Interestingly, Motor1 was able to bind the cancer drug paclitaxel and increase its solubility in water by a factor of 2000. Finally, bioactivity assays showed that the increase in solubility by paclitaxel via Motor1 led to a more efficient killing of the cervical cancer cell line HeLa. Conclusion. Our study reveals very low toxicity of a novel member of the cucurbit[n]uril family of nanocontainers. It demonstrates the increase in solubility of four commercially available drugs by the containers by factors ranging from 400-2700fold. Importantly, the increase in solubility of paclitaxel led to increased killing of cancer cells in vitro. These results provide initial proof-of-concept towards the use of CB[n] molecular containers as an advanced drug delivery system.

Citation Format: Gaya Hettiarachchi, Da Ma, Duc Nguyen, Lyle Isaacs, Volker Briken. Toxicology and drug delivery by novel Cucurbit[n]uril-type molecular containers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4347. doi:10.1158/1538-7445.AM2013-4347
Abstract 4348: Antitumor activity of Tivantinib (ARQ 197) is due to inhibition of tubulin polymerization in addition to c-Met inhibition.
Authors: Katayama Ryohei , Aoyama Aki , Yamori Takao , Qi Jie , Ohara Tomoko , Song Youngchul , Engelman Jeffrey A. , Fujita Naoya
Published: August 2014

The receptor tyrosine kinase c-Met is the high-affinity receptor for the hepatocyte growth factor (HGF). The HGF/c-Met axis is often dysregulated in tumors. Such c-Met activation is caused by MET gene amplification, activating mutations, and auto- or paracrine mechanisms. Thus, c-Met inhibitors are under development as anti-cancer drugs. Tivantinib (ARQ 197) was reported as a small molecule Met inhibitor and early clinical studies suggest anti-tumor activity. To assess if the anti-tumor activity of tivantinib was due to inhibition of c-Met, we investigated the activity of tivantinib in both c-Met addicted and non-addicted cancer cells for cell viability. As expected, we observed that other Met inhibitors, crizotinib and PHA-665752, suppressed the growth of c-Met addicted cancers, but not the growth of c-Met non-addicted cells. In contrast, tivantinib inhibited cell viability similarly in both c-Met addicted and non-addicted cells. These results suggest that tivantinib exhibits its antitumor activity in a c-Met status independent manner. Tivantinib treatment increased the number of G2/M phase arrested EBC1 cells similarly as vincristine treatment, whereas PHA-665752 or crizotinib treatment markedly induced cell cycle arrest at the G0/G1 phase. To identify the additional molecular target of tivantinib, we performed COMPARE analysis, in silico screening using extensive drug sensitivity database of 39 cancer cell lines (JFCR39), and identified microtubule as a target of tivantinib. Tivantinib treated cells demonstrated typical microtubule disruption similar to vincristine. In vitro microtubule polymerization assay clearly demonstrated that tivantinib inhibited microtubule assembly. From these results, tivantinib inhibits microtubule polymerization in addition to inhibiting c-MET.

Citation Format: Ryohei Katayama, Aki Aoyama, Takao Yamori, Jie Qi, Tomoko Ohara, Youngchul Song, Jeffrey A. Engelman, Naoya Fujita. Antitumor activity of Tivantinib (ARQ 197) is due to inhibition of tubulin polymerization in addition to c-Met inhibition. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4348. doi:10.1158/1538-7445.AM2013-4348
Abstract 4352: Down regulation of retinoblastoma protein expressionimpedes the antileukemic activity of erufosine.
Authors: Zaharieva Maya M. , Kirilov Milen , Chai Minquang , Berger Stefan M. , Konstantinov Spiro M. , Berger Martin R.
Published: August 2014

Erufosine is a new antineoplastic agent from the class of alkylphosphocholines, which interferes with AKT phosphorylation, the related signal transduction and confers apoptosis. An improved understanding of its mode of action is important for its potential clinical application. The present study demonstrates that erufosine's antileukemic efficacy is related to the level of cellular retinoblastoma (Rb) protein expression.

Stable Rb-knockdown in SKW-3 leukemia T-cells was induced by lentiviral delivery of constructs expressing short-hairpin-RNA species with 21 or 27 bp lengths. The cytotoxicity of erufosine was tested via MTT proliferation test on thus engineered cell populations with reduced Rb expression levels. Likewise, the clonogenicity of these engineered cell populations was determined by CFU assay before and after treatment with erufosine. The effects on cell cycle were investigated by FACS analysis. The expression of signal transduction factors was examined by RT-PCR and immunoblot.

The Rb protein expression levels in the shRNA-transduced cells ranged from 1%- 36% of wild type or nonsense control. Low Rb expression correlated with significantly diminished antiproliferative and anticlonogenic activities of erufosine (p<0.05) at concentrations near and above the IC50. Erufosine induced a G2 arrest, and complete Rb-deficiency aggravated this condition. With regard to proteins involved in cell cycle and apoptosis induction, Rb-deficiency reduced the expression of cyclin D3, that of the subsequently regulated kinase Cdk 4 and activated caspases 3 and 9, followed by PARP-cleavage. In partial variance, erufosine reduced cyclin D3 at RNA and protein levels, but affected not or only slightly the expression of its partner kinase Cdk 4.

Rb-knockdown impaired the cytotoxic effect of erufosine significantly and contributed to the formation of a resistant cell fraction. It is concluded that

Rb is a main mediator of erufosine's antileukemic activity and this suggests that the effect of the drug in patients might be predicted by determining the Rb expression status.

Citation Format: Maya M. Zaharieva, Milen Kirilov, Minquang Chai, Stefan M. Berger, Spiro M. Konstantinov, Martin R. Berger. Down regulation of retinoblastoma protein expressionimpedes the antileukemic activity of erufosine. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4352. doi:10.1158/1538-7445.AM2013-4352
Abstract 4369: Compound 1, a quinoxaline-based small molecule, regulates the cancer cell proliferantion by the down-regulation of maternal embryonic leucine zipper kinase gene expression in brain tumor cells.
Authors: Choi Sang-Un , Park Chi Hoon , Ahmed Mansoor M. , Yoo Stephen , Park Shin-Young , Kim Kwangrok
Published: August 2014

Maternal embryonic leucine zipper kinase (MELK), a member of the snf1/AMPK family of serine-threonine kinase, was indicated in stem cell renewal, cell cycle progression. Recently, several groups reported that MELK expression level is highly correlated with malignancy of a variety of tumors and its expression in the cancer cells is a negative factor of radiotherapy in cancer patients. Therefore, MELK can be a good target for cancer drug development. Previously, we tested the neuronal cell differentiation by the compound-1, a quinoxaline-based small molecule, with rat mesenchymal stem cells, and we found that the gene expression level of MELK is dramatically decreased in the differentiated cells in comparison with the parental stem cells. Based on this phenomenon, we checked the MELK gene expression in the several central nerve system cancer cell lines such as U87MG, XL498 and SNB19, and examined the change of the gene expression level by treatment of compound 1. All the cancer cells tested in this study expressed the MELK gene and its expression level is significantly decreased by the treatment of 20 uM compound 1. To confirm the relationship between the MELK gene expression and cell proliferation in brain tumor cells, we transfected the MELK gene to the U87MG cells and tested the cell growth rate in the presence or absence of compound 1 in comparison with parental cells. The cell growth rate of MELK gene-transfected cancer cells increased in comparison with the non-transfected control cells, and its overexpression can recover the down-regulation of the cell proliferation by treatment of compound 1. Meanwhile, the treatment of MELK gene siRNA decreased the cell proliferation rate of the U87MG cells in time dependent manner. In the cell cycle analysis, compound 1 induced both the increase of apoptotic cell rate from less than 5% to 22% and the decrease of G0/G1 phase U87MG cells in comparison with control. Meanwhile, the S and G2/M phase is not changed by the treatment of compound 1 in the U87MG cells. These results support MELK has an effect on regulation of cancer cell proliferation, and it may be a good target for cancer drug development.

Citation Format: Sang-Un Choi, Chi Hoon Park, Mansoor M. Ahmed, Stephen Yoo, Shin-Young Park, Kwangrok Kim. Compound 1, a quinoxaline-based small molecule, regulates the cancer cell proliferantion by the down-regulation of maternal embryonic leucine zipper kinase gene expression in brain tumor cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4369. doi:10.1158/1538-7445.AM2013-4369
Abstract 4378: Poly(ADP-ribose)glycohydrolase (PARG) is a novel therapeutic target in breast cancer.
Authors: Lovato Amanda , Zhao Tiejun , Sun Qiang , Witcher Michael
Published: August 2014

Poly(ADP-ribose)polymerase (PARP) is an enzyme responsible for catalyzing the addition of poly(ADP-ribose) chains onto target proteins in a process known as PARylation. This post-translational modification has been implicated in numerous cellular processes including gene expression, chromatin remodeling, apoptosis and DNA damage repair. In particular, the role of PARP activation in response to DNA damage has been intensely studied. BRCA mutated tumors are highly sensitive to DNA breaks and are therefore critically dependent on alternative repair mechanisms involving PARP. This forms the basis for a synthetic lethal therapeutic approach that has been met with much success in the clinic. BRCA mutated breast and ovarian tumors, in particular, are acutely sensitive to PARP inhibitors. Currently, it is unclear from in vitro and clinical studies to what extent PARP inhibitors can be used as chemotherapeutics to treat tumors beyond those harboring BRCA mutations. Due to the insensitivity of most solid tumors to PARP inhibitors, we have begun to study the role of the dePARylating enzyme poly(ADP-ribose)glycohydrolase (PARG), as a possible target in cancer therapy. PARG, like PARP, maintains a critical role in DNA repair and can therefore be a target in cells with defective repair mechanisms (e.g. BRCA mutant cells). We further hypothesize that targeting this factor may activate the expression of tumor suppressor genes, thus limiting cell proliferation. Thus far, we have novel data indicating that targeting PARG may indeed have therapeutic benefit. We show that PARG knockdowns of the T47D and MDA-MB-468 breast cancer cell lines have dramatically reduced rates of cellular proliferation. Gene expression analyses of the knockdown cells indicated that cell cycle related pathways may be affected. As such, we examined cell cycle profiles by BrdU and PI staining and found that PARG knockdown cells were arrested in S phase. These results are particularly striking given that these cells show resistance to PARP inhibition. Subsequent studies using the PARG inhibitor gallotannin successfully reconstituted the results obtained with the PARG knockdowns. Overall, our data illustrates that PARG has great potential as a new anti-cancer target.

Citation Format: Amanda Lovato, Tiejun Zhao, Qiang Sun, Michael Witcher. Poly(ADP-ribose)glycohydrolase (PARG) is a novel therapeutic target in breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4378. doi:10.1158/1538-7445.AM2013-4378
Abstract 4395: Targeting ribosomal proteins for therapeutic inhibition of melanoma growth.
Authors: Kardos Gregory R. , Robertson Gavin P.
Published: August 2014

Disruption of ribosome biogenesis leads to the activation of the RP-MDM2-p53 pathway along with other yet to be identified pathways. Ribosome biogenesis is inherently tied to protein synthesis, a process relied upon in cancer to sustain its increased proliferative properties. The ability to activate a tumor suppressor and simultaneously target a process heavily dependent upon for cancer growth is a promising therapeutic angle that deserves exploration. In the present study, we knocked down expression of all large subunit ribosomal proteins to examine their effect on melanoma growth. We found that targeting many ribosomal proteins lead to decreased cancer cell viability. siRNA mediated knocking down of RPL13, a representative ribosomal protein of this group, resulted in increased p53 expression and cell cycle arrest dependent on the RP-MDM2-p53 pathway. Furthermore, inhibition of protein synthesis in culture and decreased tumor growth by 50-90% in animals was observed upon knockdown of RPL13. We conclude that the cell cycle arrest is due in large part to the increased p53 expression caused by the binding of RPL5 and RPL11 to MDM2, however the inhibition of melanoma growth cannot be explained solely by an increase in the tumor suppressor. The accompaniment of decreased protein synthesis provides an additional mechanism contributing to decreased growth. Thus, targeting ribosomal proteins in cancer may serve as an underappreciated therapeutic treatment.

Citation Format: Gregory R. Kardos, Gavin P. Robertson. Targeting ribosomal proteins for therapeutic inhibition of melanoma growth. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4395. doi:10.1158/1538-7445.AM2013-4395
Abstract 4397: Regulation of tamoxifen sensitivity by a PAK1-EBP1 signaling pathway in breast cancer.
Authors: Awasthi* Smita , Ghosh* Arundhati , Peterson Jeffrey R. , Hamburger Anne W.
Published: August 2014

EBP1, an ErbB3 binding protein isolated in our laboratory, sensitizes breast cancer cells to tamoxifen in part by decreasing ErbB2 protein levels. The p21 regulated serine/threonine kinase PAK1, implicated in tamoxifen resistance, phosphorylates EBP1 in vitro and in vivo at T261. Phosphorylation of EBP1 at this site induces tamoxifen resistance. We thus postulated that inhibition of PAK1 activity, by restoring EBP1 function, could ameliorate the hormone refractory phenotype of ErbB2 overexpressing breast cancer cells. Transient transfection studies indicated that an EBP1 T261E mutant, which mimics EPB1 phosphorylated by PAK1, increased ErbB2 protein levels. An EBP1 T261A mutant, unable to be phosphorylated by PAK1, ameliorated PAK1-induced tamoxifen resistance, suggesting that phosphorylation of EBP1 by PAK1 contributes to tamoxifen resistance. We then tested if pharmacological inhibition of PAK1 activity might render hormone resistant cells, which endogenously overexpress PAK1 and ErbB2, tamoxifen sensitive. Transfection of EBP1 sensitized cells to tamoxifen. The T261A EBP1 mutant more potently sensitized cells to tamoxifen than wild type EBP1. IPA-3, a specific small MW PAK1 inhibitor, sensitized cells to tamoxifen only when EBP1 was ectopically expressed. However, IPA-3 did not further affect sensitivity of T261A overexperssing cells, suggesting that the phosphorylation of EBP1 by PAK1 played a role in the EBP1-induced hormone sensitivity. IPA had no effect on tamoxifen resistance in T47D cells in which EBP1 protein had been ablated by shRNA. The IPA-induced increase in tamoxifen sensitivity was accompanied by a decrease in ErbB2 levels in EBP1 overexpressing, but not vector control cells. These studies suggest that phosphorylation of EBP1 may be a novel additional mechanism of PAK1-induced hormone resistance. In addition, strategies that increase the pool of functional EBP1 and decrease PAK1 activity may affect hormone resistant cell growth. Our results also suggest that PAK1 directed therapies may be more effective in patients that express high levels of EBP1.

*contributed equally to this work

Citation Format: Smita Awasthi*, Arundhati Ghosh*, Jeffrey R. Peterson, Anne W. Hamburger. Regulation of tamoxifen sensitivity by a PAK1-EBP1 signaling pathway in breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4397. doi:10.1158/1538-7445.AM2013-4397
Abstract 4406: CARD11 is critical for in vitro and in vivo growth of ABC-DLBCL cells.
Authors: Murtie Joshua C. , Zhu Julia , Wang Hongyun , Ospina Beatriz , Cindhuchao May , Hoffman Dietmar , Greco Rita , Dorsch Marion , Adrian Francisco
Published: August 2014

Diffuse large B cell lymphoma (DLBCL) is the most common form of non-Hodgkin's lymphoma and can be divided into three distinct subtypes. The activated B cell-like (ABC) subtype is the most aggressive and is characterized by constitutive activation of the NF-κB pathway. CARD11 is a scaffolding protein and plays a critical role in the NF-κB pathway by coordinating IκB kinase activity and downstream pathway activation. Previous studies have revealed that ABC-DLBCL cell lines are dependent on the NF-κB pathway (Davis et al., 2001). Furthermore, activating mutations in CARD11 occur in approximately 10% of ABC-DLBCL patient tumors and result in constitutive activation of the NF-κB pathway (Lenz et al., 2008).

Using a combination of genetic and pharmacological tools we have further demonstrated that CARD11 is a critical node of the NF-κB pathway essential for B cell lymphoma growth in vitro and in vivo. Using a variety of pharmacological inhibitors, we demonstrate that anti-proliferative activity of each compound corresponds with its ability to inhibit the NF-κB pathway in an ABC-DLBCL cell line, TMD-8, engineered to express luciferase under the control of an NF-κB responsive promoter. Next, using doxycycline-inducible shRNAs targeting CARD11, we confirm the dependency of TMD-8 cells on CARD11 in vitro. The effects of CARD11 knockdown can be rescued by reactivation of the NF-κB pathway downstream of CARD11 by addition of TNF-α to a TMD-8 line engineered to express the TNF receptor. After validation of CARD11 dependency in vitro, we generated tumor xenografts using TMD-8 cells transduced with doxycycline-inducible shRNAs against CARD11. Upon treatment with doxycycline, TMD-8 tumor xenografts showed stasis or regression as a result of CARD11 knockdown. This inhibition of tumor growth also corresponded with significant down-regulation of the NF-kB responsive genes IL-6 and IL-10.

Together these data demonstrate that ABC-DLBCL cells and tumors require CARD11 for growth in an NF-kB dependent manner.

Citation Format: Joshua C. Murtie, Julia Zhu, Hongyun Wang, Beatriz Ospina, May Cindhuchao, Dietmar Hoffman, Rita Greco, Marion Dorsch, Francisco Adrian. CARD11 is critical for in vitro and in vivo growth of ABC-DLBCL cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4406. doi:10.1158/1538-7445.AM2013-4406
Abstract 4418: Overexpression of PGC-1α in rat cardiomyocytes protects against nucleoside reverse transcriptase inhibitor (NRTI)-induced mitochondrial toxicity.
Authors: Shim Eunwoo , Liu Daniel , Gibbons Alexander , Poirier Miriam C. , Liu Yongmin
Published: August 2014

The NRTIs Zidovudine (AZT) and Didanosine (ddI) are components of the antiretroviral combination therapies in widespread use for treatment of HIV-1 infection. However, AZT, a carcinogen in mice, is a DNA replication chain terminator that damages heart, skeletal muscle, brain and liver during long-term clinical use. In addition, both drugs are known to cause treatment-limiting mitochondrial compromise. In order to elucidate the mechanisms resulting in cardiac mitochondrial toxicity in patients using these drugs, we have evaluated rat cardiomyocyte H9c2 cells exposed for >30 passages (P) to 50 μM AZT or 50 μM AZT/50 μM ddI, doses allowing for ≥80% survival. Mitochondrial integrity, evaluated as Oxygen Consumption Rate (OCR, by Seahorse Analyzer) was reduced by ≥50% (p<0.05) in NRTI-exposed cells at most P compared to unexposed controls. Progressive abnormal mitochondrial morphology was observed, with time of exposure, by electron microscopy (EM). In addition, in AZT/ddI-exposed cells there were reductions (about 50%) in protein levels of PGC-1α, a master regulator of mitochondrial function and biogenesis, and its downstream effector NRF-1, compared to unexposed cells. Hypothesizing that up-regulation of PGC-1α might protect cells from AZT/ddI-induced mitochondrial damage, H9c2 cells were infected with lentivirus carrying PGC-1α, and shown by Western blot to overexpress PGC-1α. When both the H9c2 cells and the H9c2/ PGC-1α cells were exposed to 50 μM AZT/50 μM ddI, significantly-increased (20-50%) OCR levels were observed at P 2, 6, 8, 12 and 15 in the H9c2/ PGC-1α cells, compared to the H9c2 cells (p<0.05 for all P). In addition, Western blot showed about 50% higher levels of NRF-1 in AZT/ddI-exposed H9c2/ PGC-1α cells, compared to AZT/ddI-exposed H9c2 cells. These studies support our hypothesis that PGC-1α overexpression protects cardiomyocytes from NRTI-induced mitochondrial compromise, and suggest that the AZT/ddI-induced reduction in protein levels of PGC-1α is a major mechanism contributing to AZT/ddI induced toxicity. We plan to search for natural products that offer similar protection and evaluate their effect on genotoxicity and mitochondrial toxicity of the antiretroviral NRTIs.

Citation Format: Eunwoo Shim, Daniel Liu, Alexander Gibbons, Miriam C. Poirier, Yongmin Liu. Overexpression of PGC-1α in rat cardiomyocytes protects against nucleoside reverse transcriptase inhibitor (NRTI)-induced mitochondrial toxicity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4418. doi:10.1158/1538-7445.AM2013-4418
Abstract 442: CD10 positive head and neck squamous cell carcinoma have chemoresistant and radioresistant properties.
Authors: Fukusumi Takahito
Published: August 2014

Background: Despite recent advances in the understanding of head and neck squamous cell carcinoma (HNSCC) and the development of novel therapeutic targets, five-year survival rates for HNSCC have not improved in more than three decades. Prognosis for recurrent or metastatic HNSCC remains poor. Thus, it is important for improvement of HNSCC therapy, to research therapeutic resistant mechanism. We found CD10 positive HNSCC cells have chemoresistant and radioresistant properties.

Materials and Methods: In order to investigate HNSCC chemoresistant and radioresistant properties, we used Lyoplate (company:BD).This reagent contains 242 purified monoclonal antibodies to cell surface markers and can be used for screening cells with flow cytometry. First, in three HNSCC cell lines, we compared cell surface antigen expression patterns to normal cultured cells, temporarily cisplatin exprosured cells and irradiated cells.

Result: We found four cell surface markers that upragulated in cisplatin exprosured cells and irradiated cells. These are CD10, CD15s, CD146, CD282. Next, we compered these expression pattern to normal cultured cells and cisplatinresistant cells. CD10 and CD282 was upregulated. Focused on CD10, we performed cisplatin MTT assay. CD10 positive cells had more resistant property than CD10 negative cells.

Conclusion: CD10 is a cell surface zinc-dependent metalloendopeptidase, which is expressed in a wide variety of normal cells. CD10 is also expressed by several solid tumors such as melanoma and several carcinomas originating from kidney, lung, skin, pancreas, prostate, liver, breast, stomach, cervix, and bladder. Several studies have shown that CD10 might be a good marker for differentiating between primary tumors and derived metastases, and therefore for evaluating tumor progression. In HNSCC, only one report demonstrated role of CD10 in the differentiation and growth of neoplastic cells and concluded that CD10 expression can have an important role in the tumor invasion, metastases. Our date is first report that CD10 positive HNSCC have chemoresisitant and radioresistant properties. This result may correlate with CD10 positive HNSCC has invasive and metastatic character.

Learning Objective: We identified CD10 positive HNSCC have chemoresisitant and radioresistant properties.

Citation Format: Takahito Fukusumi. CD10 positive head and neck squamous cell carcinoma have chemoresistant and radioresistant properties. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 442. doi:10.1158/1538-7445.AM2013-442
Abstract 4438: Gold nanoparticle imaging and radiotherapy of brain tumors in mice.
Authors: Hainfeld James F. , O'Connor Michael J. , Slatkin Daniel N. , Dilmanian F Avraham , Smilowitz Henry M.
Published: August 2014

Aims: To test intravenously injected gold nanoparticles for X-ray imaging and radiotherapy enhancement of large, imminently lethal intracerebral malignant gliomas. Materials & Methods: ∼11nm gold nanoparticles were injected intravenously and brains imaged by microCT. Fifteen hours after an iv dose of 4 g Au/kg, brains were irradiated with 30 Gy 100kVp X-rays. Results: Gold uptake gave a 19:1 tumor-to-normal brain ratio with 1.5% w/w gold in tumor, calculated to increase local radiation dose by ∼300%. Mice receiving gold and radiation (30 Gy) showed 50% long-term (> 1 year) tumor-free survival, whereas all mice receiving radiation only died. In a second experiment using 35 Gy, long-term survival was 56% with AuNPs plus radiation versus 18% with radiation alone. Combining both experiments, the radiation plus AuNP treatments were statistically distinct from the radiation-only groups (p = 0.014). Conclusions: Intravenously injected gold nanoparticles cross the blood-tumor barrier but are largely blocked by the normal blood brain barrier, enabling high-resolution CT tumor imaging. Gold radiation enhancement significantly improved long-term survival compared to radiotherapy alone. This approach holds promise to improve therapy of human brain tumors and other cancers.

Citation Format: James F. Hainfeld, Michael J. O'Connor, Daniel N. Slatkin, F Avraham Dilmanian, Henry M. Smilowitz. Gold nanoparticle imaging and radiotherapy of brain tumors in mice. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4438. doi:10.1158/1538-7445.AM2013-4438
Abstract 4440: Identification and characterization of small molecule inhibitors that radiosensitize K-RAS mutant rectal cancers.
Authors: Kleiman Laura B. , Krebs Angela , Kim Stephen , Hong Theodore S. , Haigis Kevin M.
Published: August 2014

Around 40% of rectal cancers harbor activating K-RAS mutations, and these mutations are associated with resistance to chemoradiotherapy. We aimed to identify small molecule inhibitors (SMIs) that synergize with ionizing radiation (IR) (“radiosensitizers”) that could be incorporated into current treatment strategies for K-RAS mutant locally advanced rectal cancer (LARC) patients. We first optimized a high-throughput assay for measuring individual and combined effects of SMIs and IR that produces similar results to the gold standard colony formation assay. Using this screening platform and K-RAS mutant rectal cancer cell lines, we tested SMIs targeting diverse signaling pathways for radiosensitizing activity and then evaluated our top hits in follow-up experiments. The two most potent radiosensitizers were the Chk1/2 inhibitor AZD7762 and the PI3K/mTOR inhibitor BEZ235. The chemotherapeutic agent 5-fluorouracil (5-FU), which is used to treat LARC, synergized with AZD7762 and enhanced radiosensitization by AZD7762 but not by BEZ235. This study is the first to compare different SMIs in combination with IR for the treatment of K-RAS mutant rectal cancer, and our findings suggest that Chk1/2 inhibitors should be evaluated in new clinical trials for LARC.

Citation Format: Laura B. Kleiman, Angela Krebs, Stephen Kim, Theodore S. Hong, Kevin M. Haigis. Identification and characterization of small molecule inhibitors that radiosensitize K-RAS mutant rectal cancers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4440. doi:10.1158/1538-7445.AM2013-4440