bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2112: The role of reactive oxygen species (ROS) in cell death induced by thymidylate synthase inhibitors.
Authors: Ozer Ufuk , Barbour Karen W. , Berger Franklin G.
Published: August 2014

Thymidylate synthase (TS) catalyzes the reductive transfer of a methyl group from N5,N10-methylenetetrahydrofolate (CH2H4PteGlu) to dUMP, forming dTMP and dihydrofolate (H2PteGlu). It is generally accepted that inhibition of the enzyme leads to a deficiency of dTMP, followed by genome damage and programmed cell death. As such, TS has long been viewed as an important target of anti-neoplastic agents, such as 5-fluorouracil (FUra) and ratitrexed (RTX), which function as inhibitors of the enzyme. Oxidative stress is well-recognized as having a central role in cellular response to a number of DNA damaging agents, and may be a proximate cause of therapy-induced cell death. Despite such recognition, little detail exists on the origin and regulation of reactive oxygen species (ROS) during TS-directed chemotherapy. We have undertaken a detailed examination of the origin, nature, and role of ROS in cell death mediated by TS inhibitors. In colon tumor cell line HCT116, fluorescence detection of H2O2 and O2•− (using H2DCFDA and DHE, respectively) showed that profound increases in ROS levels occur during drug exposure. These increases are attenuated by treatment with the antioxidant N-acetylcysteine (NAC), by addition of thymidine to the medium and by TS overproduction, indicating that it is indeed TS inhibition and the resulting dTMP deficiency that is responsible. Oxidative damage to DNA, as indicated by 8-oxo-guanine formation, is elevated in drug-treated cells. Apoptotic indices, as measured by TUNEL assays, parallel changes in ROS levels, i.e., they are induced in response to drug, and are inhibited by NAC, thymidine, and TS overproduction. We have identified NADPH oxidase (NOX) as the primary source of increased ROS following exposure to TS-directed agents, as indicated by observing decreased ROS production apoptotic indices in cells treated with NOX inhibitors. We have found that in response to TS inhibitors, NOX enzyme activity increases in association with induction of the transcript for p67phox, which specifically regulates the NOX2 isoform. These effects were reduced by thymidine and by TS overproduction. Based on these results, we conclude that augmentation of NOX2 activity via induction of p67phox mRNA expression is the proximate cause of programmed cell death elicited by dTMP deficiency.

Citation Format: Ufuk Ozer, Karen W. Barbour, Franklin G. Berger. The role of reactive oxygen species (ROS) in cell death induced by thymidylate synthase inhibitors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2112. doi:10.1158/1538-7445.AM2013-2112
Abstract 2113: Arsenic hexoxide inhibits NF-κB activity through, at least in part, suppression of IκBα phosphorylation in MCF-7 human breast cancer cells.
Authors: Lee Won Sup , Kim Min Jeong , Jung Ji-Hyun , Yun Jeong Won , Lu Jing Nan , Yi Sang mi , Kim Hye Jung , Chang Seong-Hwan , Kim GonSup , Hong Soon Chan , Hwa Woo song
Published: August 2014

Arsenic hexoxide (As4O6) has been used in Korean folk remedy for the treatment of cancer since the late 1980’s. Evidence suggests that the anti-cancer effects of As4O6 are different from those of As2O3. Nuclear factor-κB (NF-κB) is a well-known transcription factor involved in cell proliferation, invasion and metastasis. Here, we investigated the effects of As4O6 on NF-κB activity, NF-κB-regulated gene expressions, and NF-κB-mediated cellular responses. As4O6 suppressed NF-κB activation induced by TNF-α, and the some of the downstream NF-κB-regulated proteins involved in caner proliferation, metastasis and anti-apoptosis. As4O6 also suppressed IκB phosphorylation in a time-dependent manner, suggesting the suppression of NF-κB results from at least in part by inhibiting IκB degradation. We also confirmed the anti-NF-κB activity of As4O6 with synergism with TNF-α at the molecular levels. This study demonstrates that As4O6 inhibits NF-κB activation and NF-κB regulated proteins at least in part through the inhibition of IκB phosphorylation. [This study was supported by a grant from the National R&D Program for Cancer Control, Ministry for Health, Welfare & Family Affairs, Republic of Korea. (0820050), and National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (20120002631).]

Keywords: Arsenic hexoxide, Nuclear factor-κB, IκB, cancer

Citation Format: Won Sup Lee, Min Jeong Kim, Ji-Hyun Jung, Jeong Won Yun, Jing Nan Lu, Sang mi Yi, Hye Jung Kim, Seong-Hwan Chang, GonSup Kim, Soon Chan Hong, Woo song Hwa. Arsenic hexoxide inhibits NF-κB activity through, at least in part, suppression of IκBα phosphorylation in MCF-7 human 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 2113. doi:10.1158/1538-7445.AM2013-2113
Abstract 2131: Functional assays to define agonists and antagonists of sigma-2 receptors.
Authors: Mach Robert H. , Zeng Chenbo , Rothfuss Justin M. , Zhang Jun , Vangveravong Suwanna , Chu Wenhua , Tu Zhude , Li Shihong
Published: August 2014

The sigma receptor was originally defined pharmacologically as the specific binding site for a group of compounds named as sigma ligands. The sigma-2 receptor has been identified as a biomarker in proliferating tumors. It regulates cell growth and is an emerging target for cancer diagnosis and therapeutics. Up to date numerous sigma-2 selective ligands have been developed. However, there is no well-established functional assay for defining agonist/antagonist for sigma-2 receptors. Many sigma-2 ligands with diverse structures have been shown to induce cytotoxicity in a variety of cancer cells by triggering caspase-independent and caspase-dependent apoptosis. Therefore, in the current study, we propose to use cell viability and caspase 3 activity, a hallmark of apoptosis, as functional assays to define agonist/antagonist for sigma-2 receptors. Three classes of sigma-2 ligands developed in our laboratory were tested in these two assays in two tumor cell lines: mouse breast cancer cell line EMT6 and human melanoma cell line MDA-MB435. Based on the ratios of the EC50 values of our sigma-2 ligands to the EC50 of siramesine, we categorized the azabicyclononane analogs as agonists (e.g. SV 119) or partial agonists (e.g. WC 26 and SV 95), and benzamide analogs as non-canonical antagonists (e.g. RHM-1) since they do not trigger cell death, and they do not block sigma-2 agonist-induced cell death. SV 95 partially blocked SV 119-induced cytotoxicity. The establishment of functional assays for defining agonist/antagonist will facilitate functional characterization of sigma-2 ligands and sigma-2 receptors.

Citation Format: Robert H. Mach, Chenbo Zeng, Justin M. Rothfuss, Jun Zhang, Suwanna Vangveravong, Wenhua Chu, Zhude Tu, Shihong Li. Functional assays to define agonists and antagonists of sigma-2 receptors. [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 2131. doi:10.1158/1538-7445.AM2013-2131
Abstract 2140: Oral antiangiogenic therapy suppresses xenograft growth and is well tolerated in rats.
Authors: Benny Ofra , Bazinet Lauren , D'Amato Robert
Published: August 2014

Lodamin, the oral formulation of TNP-470, a methionine aminopeptidase 2 (MetAp2) inhibitor, was previously shown to significantly inhibit angiogenesis and suppress the progression of fast growing murine tumors. Lodamins’ unique structure as a polymer micelle composed of poly(ethylene-glycol)-poly(lactic)acid (PEG-PLA) conjugate of TNP-470 is pharmacologically advantageous over the non-conjugated form. It shows better stability, oral availability, solubility in water and importantly, has an improved safety profile (Benny O. Folkman J et al, Nat Biotech, 2008). We now report the successful large scale synthesis of Lodamin, yielding pure and active product composed of mPEG-PLA-TNP-470 conjugate without residual free drug as confirmed by NMR and LC-MS/MS. The safety profile of Lodamin was studied in an acute high dose rat study (4 fold higher than therapeutic dose). Lodamin was administered at 10mg/kg or 60mg/kg via oral gavage while control rats were given vehicle only. After 7 days of daily treatment, no changes in body weight or behavior were observed compared to the control group. On day 8 post treatment, gross pathology of all rats was examined. This data showed Lodamin treatment had no adverse effect on tissue morphology. Lodamin is a potent antiangiogenic drug in numerous in vivo murine models including the corneal micropocket assay, matrigel assay, Delayed-Type Hypersensitivity (DTH) reaction and the choroidal neovascularization (CNV) model (Benny O. Folkman J et al, Nat Biotech, 2008, Benny O. D'Amato R et al, Plos One 2010). We now show that in addition to its dramatic anti-cancer effects on Lewis lung carcinoma and B16F10 melanoma murine tumors, Lodamin suppresses human tumors induced in nude mice. The volume of subcutaneous (s.c) human glioblastoma (U87MG) xenografts were reduced by 70% after 30 days, and 71% after 45 days when administered by gavage or in drinking water, respectively. Human breast tumor (MDA-MB231, s.c) growth was inhibited by 62% after 47 days of administration, and Human hepatocarcinoma (HepG2, s.c) tumor growth was inhibited by 60% after only 12 days. Taken together we have established Lodamin as a potent antiangiogenic drug which retains TNP-470 activity without TNP-470-associated side effects. Lodamin is a safe and efficacious drug in our animal models and hopefully will be reintroduced for the treatment of cancer in patients.

Citation Format: Ofra Benny, Lauren Bazinet, Robert D'Amato. Oral antiangiogenic therapy suppresses xenograft growth and is well tolerated in rats. [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 2140. doi:10.1158/1538-7445.AM2013-2140
Abstract 2148: Novel therapy of liver metastases of cancer using Abraxane loaded into Multi-Stage Nanovector.
Authors: Tanei Tomonori , Ferrari Mauro , Godin Biana , Yokoi Kenji
Published: August 2014

Purpose; To investigate whether the Abraxane loaded within Multi-Stage Nanovector (MSV) system (MSV-ABX) is more effective in therapy of liver metastases of breast and colorectal cancer as compared to Abraxane (ABX).

Experimental Design; MTT assay was performed to evaluate sensitivity of 4T1, murine breast cancer cells and CT26, murine colon cancer cells to ABX or MSV-ABX in vitro. Experimental liver metastases were produced by intra-splenic injection of 4T1 or CT26 cells in Balb/C mice. The mice bearing liver metastases were treated with intravenous injection of (1) control (PBS), (2) ABX or (3) MSV-ABX. Then the mice were sacrificed and liver weight was measured to evaluate the therapeutic efficacy. To evaluate the biological the effects of therapeutics, we performed histological and immunohistochemical analysis of the tumor specimens. Apoptosis of 4T1 and CT26 tumor cells were evaluated by TUNEL staining, and proliferation of the tumor cells were stained using antibody to Ki67. Tumor diameter was measured in H&E stained sections.

Results; There was no significant difference in the sensitivity of 4T1 and CT26 cancer cells to ABX and MSV-ABX, in vitro. All the mice injected with 4T1 or CT26 cells developed multiple liver metastases. The liver weight was significantly lower in the mice treated with MSV-ABX as compared to those in the other groups. The tumor diameters in the liver were significantly smaller in the mice treated with MSV-ABX as compared to those in the other two groups. Immunofluorescent analysis revealed that MSV-ABX inhibited proliferation and increased apoptosis of 4T1 and CT26 tumor cells in the liver significantly more than ABX.

Conclusions; These studies demonstrate that MSV-ABX is more effective than ABX in therapy of the liver metastasis of breast and colorectal cancer in our mouse models. These data can be translated into the clinic for planning novel clinical trials using MSV-ABX.

Citation Format: Tomonori Tanei, Mauro Ferrari, Biana Godin, Kenji Yokoi. Novel therapy of liver metastases of cancer using Abraxane loaded into Multi-Stage Nanovector. [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 2148. doi:10.1158/1538-7445.AM2013-2148
Abstract 2155: Achieving complete response to locoregional disease without toxicity using temperature-sensitive liposomes and ultrasound-mediated hyperthermia.
Authors: Kheirolomoom Azadeh , Lai Chun-Yen , TAM Sarah M. , Even Lisa M. , Ingham Elizabeth S. , Fite Brett Z. , Watson Katherine D. , Ferrara Katherine W.
Published: August 2014

By stabilizing doxorubicin within temperature-sensitive liposomes and repeating delivery twice per week for four weeks, extended survival and complete response were obtained in syngeneic murine breast tumors. Further, with the stabilized formulation, cardiac toxicity and leukopenia were not detected. Liposomes composed of DPPC:MPPC:DSPE-PEG2k, 86:10:4 were prepared in the presence of copper(II) gluconate and triethanolamine and extruded with 100 nm membrane filters. Cu-liposomes were loaded with Dox at 0.2 mg-drug/mg-lipid with 100% loading. We studied the efficacy of CuDox liposomes (CuDox-LTSLs) using the highly invasive neu deletion (NDL) tumor. Treatment began when the tumor diameter reached 4 mm. The fluorescence of circulating doxorubicin in blood was quenched after the digestion of liposomes with Triton X-100, indicating that the circulating drug remained associated with copper. Dox fluorescence was restored upon reducing the pH using a citrate-saline buffer.

Over the 28 days of the study, CuDox-LTSLs were administrated intravenously two times per week at a therapeutic level of 6 mg-drug/kg-body weight. The entire tumor was insonified with a peak ultrasound pressure of 1.1 MPa at a frequency of 1.5 MHz at 42°C for 5 min prior to and 20 min post drug injection. A total of 30 mice were studied, including groups spanning drug treatment with ultrasound, ultrasound, drug treatment and no treatment. Although a single dose administration of CuDox-LTSLs combined with insonation of the entire tumor suppressed the tumor growth, complete response was achieved only upon repeated treatment over a period of 28 days. At 30 days after the last administered dose, none of the mice from the other groups survive; however, 100% of mice treated with ultrasound combined with CuDox-LTSLs survive and the remaining tumor is undetectable. In conclusion, repeated treatment of stabilized temperature sensitive doxorubicin liposomes is highly effective in the treatment of aggressive murine tumors.

Acknowledgement: NIHR01CA134659 and NIHR01CA103828

Citation Format: Azadeh Kheirolomoom, Chun-Yen Lai, Sarah M. TAM, Lisa M. Even, Elizabeth S. Ingham, Brett Z. Fite, Katherine D. Watson, Katherine W. Ferrara. Achieving complete response to locoregional disease without toxicity using temperature-sensitive liposomes and ultrasound-mediated hyperthermia. [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 2155. doi:10.1158/1538-7445.AM2013-2155
Abstract 2156: Targeting polo-like kinase 1 with novel inhibitors of the polo-box domain.
Authors: Baxter Merissa , Craig Sandra , McInnes Campbell , Wyatt Michael D.
Published: August 2014

Polo-like Kinase 1 (PLK1) is a central player in cell cycle regulation and mitotic progression. Observations that PLK1 is over-expressed in many cancer types have led to efforts to target this protein therapeutically. Several ATP-binding site inhibitors of PLK1 have advanced to clinical trials, but these compounds inhibit at least three of the four known mammalian PLKs to varying degrees. This is problematic because, for example, PLK3 acts as a tumor suppressor and has opposing functions to PLK1. Moreover, a recent study identified a single point mutation near the ATP binding site that renders PLK1 resistant to several structurally unrelated ATP-based inhibitors, while retaining kinase activity. The Polo-box domain (PBD) of each PLK is a phospho-peptide binding motif that determines substrate recognition and subcellular localization. Our approach is to target the PBD of PLK1 to achieve desired PLK1 selectivity and improve efficacy. We recently reported a structure-activity relationship for a series of fragment-ligated inhibitory peptides (FLIPs) designed to be selective for the PBD of PLK1 (Mol. Cancer Ther., 2012, 11, 1683-1692). Our most potent compound in that class possessed a binding affinity to the PLK1 PBD (IC50 value of 8.6 μM), and insignificant levels of PLK3 binding up to 600 μM, which represents at least a 100-fold selectivity. The compound was able to induce cellular phenotypes consistent with a PLK1 knockdown, including aberrant mitoses and induction of apoptosis. Here, we report the development of new FLIP analogues with improved potency in binding to the PLK1 PBD. Specifically, we have designed, synthesized, and tested new benzoic acid capping groups to exploit a hydrophobic cleft in the PBD binding site. This newer generation of compounds possess binding affinities to the PLK1 PBD with IC50 values below 1 μM. Studies are in progress to determine PLK1 selectivity and activity in cells. Future studies will explore the ability of PBD-targeted compounds to circumvent resistance to ATP-based compounds.

Citation Format: Merissa Baxter, Sandra Craig, Campbell McInnes, Michael D. Wyatt. Targeting polo-like kinase 1 with novel inhibitors of the polo-box domain. [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 2156. doi:10.1158/1538-7445.AM2013-2156
Abstract 2167: Strengthening anti-cancer effects on non-small cell lung carcinoma by dual blockage of MET and EGFR in a context-dependent manner.
Authors: Zhang Yu-Wen , Staal Ben , Essenburg Curt , Lewis Steven , Kaufman Dafna , Vande Woude George F.
Published: August 2014

Non-small cell lung carcinoma (NSCLC) accounts for more than 80% of lung cancer, the leading cause of death among all cancer casualties. The MET receptor tyrosine kinase (RTK) is often co-expressed with EGFR in the NSCLC cells, and is an important alternative resistant mechanism for escaping EGFR-targeted therapy. To assess the benefit of dual blockage of MET and EGFR for NSCLC therapy, we investigated the activities of these two RTK pathways in several NSCLC cell lines carrying distinct cellular contexts, and determined their responses to SGX523 and erlotinib, the small molecule kinase inhibitors of MET and EGFR, respectively. Here, we showed that MET can cross-activate EGFR in MET-amplified or overexpressing cells via hetero-receptor dimerization, and the MET-dependent phosphorylation of EGFR can be abolished by SGX523 but not by erlotinib. More importantly, combined inhibition of MET and EGFR in vitro results in a maximal suppression of downstream ERK and AKT activation and of cell proliferation when their ligands (HGF and EGF) are present. Furthermore, we demonstrated that SGX523 and erlotinib combination strengthens anti-cancer activity in vivo in a cellular context-dependent manner. The combination led to regression of MET-amplified H1993 tumors by enhancing suppression of proliferation and inducing apoptosis, whereas significantly reduced MET-non-amplified H1373 tumor growths by suppression of proliferation without inducing apoptosis. Although SGX523 alone was sufficient to achieve near complete regression of MET-addicted EBC-1 tumors, its combination with erlotinib strongly inhibited viability of a population of SGX523-insensitive cells emerging from SGX523-treated EBC-1 tumor relapse. Our data suggest that dual blockage of MET and EGFR can enhance anti-cancer effects on NSCLC in a context-dependent manner, and provide a strong rationale and mechanisms for combining MET and EGFR inhibitors for NSCLC therapy.

Citation Format: Yu-Wen Zhang, Ben Staal, Curt Essenburg, Steven Lewis, Dafna Kaufman, George F. Vande Woude. Strengthening anti-cancer effects on non-small cell lung carcinoma by dual blockage of MET and EGFR in a context-dependent manner. [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 2167. doi:10.1158/1538-7445.AM2013-2167
Abstract 2201: The molecular signature of tumor cells that are sensitives to a new tight binder NAMPT inhibitor.
Authors: Hilairet Sandrine , Arigon Jerome , Nardi Frederico , Jegham Samir , Peleraux Annick , Cosnier Sylvie , Floutard Regine , Jbilo Omar , August Paul , Shomali Maysoun , Autelitano Francois , Arrebola Rosalia , Reiling Stephan , Pollard Jack , Adrian Francisco , Sager Jason , Lengauer Christoph , Dorsh Marion , Garcia-Echeverria Carlos , Bouaboula Monsif
Published: August 2014

SAR154782 is a tight binder, low reversible NAMPT inhibitor that has been identified by a phenotypic screen and chemogenomic approaches. This class of NAMPT inhibitor induces a profound and time dependent modulation of tumor metabolism which in turn induces the reprogrammation of tumor cell transcriptome. The transcriptional profile revealed that different metabolic and oncogenic signaling pathways are modulated by SAR154782. In order to define the context of tumor cell sensitivity to NAMPT inhibition, the effects of this compound on tumor cell growth and viability have been assessed against a panel of about 240 cell lines from different tissue origin. Based on the difference of tumor cell lines sensitivity to NAMPT inhibitor, a panel of 60 genes that are differentially expressed in the sensitive versus non-sensitive cell lines has been identified. Interestingly, the low expression of NAMPT concomitant with the high expression of Sirtuin1 and PARP1 are the main outstanding features in the panel of genes identified for the most sensitive cell lines. This finding is in agreement with high NAD turnover. The relevance of the preceding biomarkers to determine the potential cancer target population that could benefit upon treatment with a NAMPT inhibitor has been investigated in in vivo settings by evaluating the antitumor activity of an advanced new class of NAMPT inhibitors in several mouse tumor xenografts and human primary tumor models.

The results obtained from these studies highlighted the modulation upon NAMPT inhibition of important tumor growth pathways and revealed for the first time a potential molecular signature of tumor sensitivity and support further investigation into use of the signature for patient stratification in the clinical setting

Citation Format: Sandrine Hilairet, Jerome Arigon, Frederico Nardi, Samir Jegham, Annick Peleraux, Sylvie Cosnier, Regine Floutard, Omar Jbilo, Paul August, Maysoun Shomali, Francois Autelitano, Rosalia Arrebola, Stephan Reiling, Jack Pollard, Francisco Adrian, Jason Sager, Christoph Lengauer, Marion Dorsh, Carlos Garcia-Echeverria, Monsif Bouaboula. The molecular signature of tumor cells that are sensitives to a new tight binder NAMPT inhibitor. [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 2201. doi:10.1158/1538-7445.AM2013-2201
Abstract 2202: Classification of DPYD variations using an isogenic system of expression.
Authors: Offer Steven M. , Wegner Natalie J. , Fossum Croix , Diasio Robert D.
Published: August 2014

The pyrimidine analog 5-fluorouracil (5-FU) was developed in the 1950s and remains one of the most widely used chemotherapy drugs in use today. A significant portion of patients who receive 5-FU suffer severe adverse toxicities, and, in extreme cases, death. Dihydropyrimidine dehydrogenase (DPD, encoded by the DPYD gene) rapidly degrades 85% of administered 5-FU, and as such, limits the amount of drug available for conversion into active metabolites. Genetic variations in DPYD have been suggested to increase the risk of 5-FU toxicity. Large-scale sequencing efforts have identified nearly 100 missense and nonsense DPYD variations, however only a handful of alleles have been studied for their association with 5-FU toxicity. Most studies have focused on a single allele (DPYD*2A), which only accounts for a small fraction of DPD deficiencies. Further complicating the interpretation of DPYD alleles, many individuals carry multiple variations within the DPYD gene. The purpose of this study was to identify missense variants that affect the enzyme function of DPD using a mammalian isogenic system of expression. Expression vectors encoding missense DPYD variations were generated by site directed mutagenesis and the expressed protein assayed for enzyme activity. Cells expressing selected variants were also teasted for sensitivity to 5-FU using a novel implementation of electrical impedance based realtime cellular analysis. The results of functional studies are compared to in silico predictions used to classify variants as deleterious or benign. An appreciable fraction of DPD variants showed enzyme activity that was significantly lower than that of wildtype protein. Within the deficient group of variants, a gradient of activities was noted, prompting the grouping of variants as mildly, moderately, and severely impaired. In addition to loss-of-function variations, a few hyperactive DPYD alleles were identified. This novel class of DPYD variants may be protective against 5-FU toxicity at the cost of treatment efficacy. The further evaluation of hyperactive alleles in clinical studies will be necessary to determine their impact on patient outcomes. Overall these data will facilitate the development of predictive tests for 5-FU toxicity by allowing researchers to prioritize variations for association studies.

Citation Format: Steven M. Offer, Natalie J. Wegner, Croix Fossum, Robert D. Diasio. Classification of DPYD variations using an isogenic system of expression. [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 2202. doi:10.1158/1538-7445.AM2013-2202
Abstract 2243: Clonogenic 3D high throughput screening in mutant KRAS dependent cancer cells - a chemogenomic approach.
Authors: Koundinya Malvika , Sudhalter Judith , Courjaud Albane , Lionne Bruno , Touyer Gaetan , Bonnet Luc , Menguy Isabelle , Schreiber Isabelle , Perrault Christelle , Vougier Stephanie , Benhamou Brigitte , Simard Daniel , Castaldi Maria Paola , Tomlinson Ronald , Reiling Stephan , Cao Hui , Harper David , Bouaboula Monsif , Pollard Jack , Grepin Claudine , Garcia-Echeverria Carlos , Adrian Francisco , Cornella-Taracido Ivan , Arrebola Rosalia , Morris Aaron J.
Published: August 2014

Activating KRAS mutations are oncogenic and prevalent in multiple tumor types, being found in as high as 90% of pancreatic carcinomas and 50% of colorectal carcinomas. In spite of its attractiveness for therapeutic intervention to date no KRAS-targeted drug therapies have been approved. Most of the reported mutant KRAS directed cellular screening efforts utilize 2-dimensional assays. Because mutant KRAS driven tumor cell lines are much more strongly dependent on activated K-Ras signaling for anchorage independent growth relative to growth when anchored to plastic, we report herein a screening strategy that leverages a 3-dimensional clonogenic growth assay in soft agar. We have performed a multi-cell line parallel phenotypic high throughput screen with our proprietary compound collection to identify pathways, targets and chemical matter with selective anti-tumor activity in mutant KRAS dependent cell lines - a Synthetic Lethal approach.

Our strategy has led to the identification of several chemical classes that inhibit the growth of multiple mutant KRAS cell lines of pancreatic and colorectal carcinoma origin, while sparing multiple wild-type KRAS cancer cell lines. Studies to elucidate their molecular mechanisms of action are underway.

Citation Format: Malvika Koundinya, Judith Sudhalter, Albane Courjaud, Bruno Lionne, Gaetan Touyer, Luc Bonnet, Isabelle Menguy, Isabelle Schreiber, Christelle Perrault, Stephanie Vougier, Brigitte Benhamou, Daniel Simard, Maria Paola Castaldi, Ronald Tomlinson, Stephan Reiling, Hui Cao, David Harper, Monsif Bouaboula, Jack Pollard, Claudine Grepin, Carlos Garcia-Echeverria, Francisco Adrian, Ivan Cornella-Taracido, Rosalia Arrebola, Aaron J. Morris. Clonogenic 3D high throughput screening in mutant KRAS dependent cancer cells - a chemogenomic approach. [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 2243. doi:10.1158/1538-7445.AM2013-2243
Abstract 2245: Overcoming cisplatin resistance by celastrol in cancer cells.
Authors: Shi Zhi
Published: August 2014

Cisplatin is one of the most widely used cytotoxic anticancer drugs for the treatment of solid malignancies, including lung, testicular, ovarian, head and neck, colorectal, bladder cancers, etc. However, its clinical efficacy is often limited by the development of chemoresistance. Several mechanisms have been implicated in cisplatin resistance, such as reduced drug uptake, increased cellular thiol/folate levels and increased DNA repair. Celastrol, a quinone methide triterpene, is an active compenent of the traditional Chinese medicinal plant Tripterygium Wilfordii, which exhibits significant antitumor activity in different cancer models in vitro and in vivo. In this study, we sought to determine the effect of celastrol on cisplatin resistance cancer cells and the possible involved molecular mechanism. Our data show that celastrol had similar cytotoxic effects on both A549 parental and A549/DDP cisplatin resistant cells. The IC50 values of celastrol to both cell lines are at the concentration of μM range. Celastrol also induced cell apoptosis with ROS accumulation, loss of mitochondrial membrane potentials, cleavage of PARP and caspases in a dose- and time-dependent manner in both cell lines. These findings might provide significant strategy for overcoming cisplatin resistance in clinic, and define the novel mechanism of celastrol-induced anticancer activity.

Citation Format: Zhi Shi. Overcoming cisplatin resistance by celastrol in 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 2245. doi:10.1158/1538-7445.AM2013-2245

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 2251: Induction of apoptosis in breast cancer cells using Kedrostis foetidissima.
Authors: Motadi Lesetja Raymond
Published: August 2014

Kedrostis foetidissima has been used by tribes throught Africa to treat various human livestock ailments. K. foetidissima, has been shown to inhibit the growth of breast cancer cell lines in vitro by induction of apoptosis in previous studies of our group1. With current chemotherapy and radiotherapy continuing to show unwanted side effects the only hope to a better safer medicinal target would include that uses medical plants that induces apoptosis with or without inducing p53 gene2,3. However, the mechanisms of action are not completely understood. In this study we are looking at the role and expressional levels of RBBp6 in breast cancer following treatment with the plant extract. In trying to achieve our objective both human breast cancer tissue and MCF7 cell lines were used to first establish the pattern of expression and then cell lines were treated with medicinal plant to evaluate the expressional patterns using both the immunohistochemistry, RTPCR and Flow cytometer.

We observed that protein as well as mRNA level of RBBP6 was inhibited by K. foetidissima in a dose-dependent manner (Fig. 1). However, in both untreated and human breast cancer tissues RBBp6 was found to be highly expressed around cells undergoing cell division. In recent studies, an apoptosis-related gene expression profiling analysis provided a clue that both p53 and bax gene were slightly upregulated at least 2 to 3 fold in response to K. foetidissima treatment in MCF7 cell2. On the other had we have reported in the past that RBBp6 acted as inhibitor of p53 gene. The findings in this study suggest that downregulation of RBBp6 may through small molecules might be a potential therapy in cancer.

Citation Format: Lesetja Raymond Motadi. Induction of apoptosis in breast cancer cells using Kedrostis foetidissima. [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 2251. doi:10.1158/1538-7445.AM2013-2251

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 2258: Celastrol shows chemopreventive properties in an inflammatory driven model for colon cancer via induction of Nrf2 transcription.
Authors: Barker Emily , Letterio John J. , Tochtrop Gregory P.
Published: August 2014

Celastrol is a natural triterpenoid isolated from the medicinal herb Triptyrgium wilfordii or Thunder of God vine. Celastrol has been shown to have a broad range of anti-inflammatory and anti-cancer activities. A mouse model for inflammatory-driven colon carcinogenesis combines a conditional T-cell deletion of the Smad4 gene with germ line deletion of the p27Kip1 gene. These genetic deletions result in spontaneous inflammation of the intestinal tract and associated early and aggressive development of cancer in the epithelia of both the small intestine and colon. In this study, administration of celastrol as a dietary supplement at a dose of 2 mg/kg is shown to be an effective strategy for cancer chemoprevention in this mouse model of colon carcinogenesis. Mice of this strain on a celastrol-supplemented diet had increased overall health and increased survival compared to mice receiving control diet. In vivo, celastrol was observed to increase the otherwise suppressed expression of cytoprotective Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) target genes (in comparison to wild type) and also to decrease expression of inducible nitric oxide synthase and accumulation of circulating nitric oxide in serum. In vitro, celastrol also induces antioxidant and cytoprotective activity through the induction of Nrf2 transcription as confirmed using macrophages derived from bone marrow of Nrf2-/- mice compared to wildtype. Celastrol induces the expression of many known Nrf2 target genes, including Hemeoxygenase-1, NAD(P)H quinone reductase-1, glutathione-S reductase, catalase and Nrf2 itself. This activity is abrogated in macrophages from Nrf2-/- mice. These data provide additional mechanistic insight on the potent anti-inflammatory activity of celastrol and present a rationale to further explore the capacity of dietary celastrol to suppress carcinogenesis in other, classical models of colon carcinogenesis with an emphasis on future potential for clinical translation.

Citation Format: Emily Barker, John J. Letterio, Gregory P. Tochtrop. Celastrol shows chemopreventive properties in an inflammatory driven model for colon cancer via induction of Nrf2 transcription. [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 2258. doi:10.1158/1538-7445.AM2013-2258
Abstract 229: Pterostilbene suppresses the generation of breast cancer stem cells within tumor microenvironment and metastasis via modulating NF-κB/microRNA448 circuit.
Authors: Yeh Chi-Tai , Chiou Jeng-Fong , Wu Chih-Hsiung , Wu Alexander TH
Published: August 2014

Tumor microenvironment plays a key role in promoting epithelial-to-mesenchymal transition (EMT), a cellular step for cancer cells to gain metastatic ability and is also associated to the generation of cancer stem cells (CSCs). Agents capable of modulating the tumor microenvironment could be more efficient in managing tumor progression. Pterostilbene, a natural stilbene isolated from blueberries have been suggested for its anti-cancer effects. Here, we explored the potential microenvironment modulating effects of pterostilbene in a M2-polarized macrophages (M2 TAMs) and breast cancer cell co-culture system. We first demonstrated that co-culturing with M2 TAMs increased the percentage of CD44+/CD24- CSC population and migratory/invasive abilities. We then showed that pterostilbene treatment dose-dependently overcame M2 TAM-induced enrichment of CSCs and metastatic potential of breast cancer cells. Mechanistically, pterostilbene suppressed NFκB, Twist1, Vimentin and increased E-cadherin expression. Importantly, pterostilbene-mediated NFκB downregulation was correlated to an increased amount of microRNA448. Finally, using non-invasive bioluminescence technique we demonstrated that pterostilbene treatment significantly suppressed M2 TAM co-cultured MDA-MB-231 tumorigenesis and metastasis. Thus, pterostilbene could be a potential anti-CSC agent for clinical development.

Citation Format: Chi-Tai Yeh, Jeng-Fong Chiou, Chih-Hsiung Wu, Alexander TH Wu. Pterostilbene suppresses the generation of breast cancer stem cells within tumor microenvironment and metastasis via modulating NF-κB/microRNA448 circuit. [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 229. doi:10.1158/1538-7445.AM2013-229
Abstract 230: Connectivity-map analyses identify FDA-approved drugs targeting breast cancer stem cells.
Authors: Bhat-Nakshatri Poornima , Goswami Chirayu , Badve Sunil , Sledge George W. , Nakshatri Harikrishna
Published: August 2014

Recently developed genomics-based tools are allowing repositioning of established drugs as cancer treatments. This approach has not been employed extensively to identify drugs that may target cancer stem cells (CSCs) of solid tumors. CD44+/CD24- cancer cells are the first described CSCs for breast cancer, which are enriched in mesenchymal stem-like (MSL) and mesenchymal-like (ML) subtypes of triple negative (TNBCs) and in anti-estrogen resistant breast cancers. We subjected gene expression array datasets containing list of genes differentially expressed in CD44+/CD24- CSCs compared to non-CSCs as well as in cells enriched for other markers of CSCs including CD271 and GD3S (six datasets in total) to connectivity map (CMAP) to identify drugs that may reverse gene expression patterns unique to CSCs. All trans retinoic acid (ATRA) was negatively connected with gene expression in CD44+/CD24-, CD271+, and GD3S+ CSCs as well as in ML and MSL subtypes of TNBCs. Independently, ATRA was found to modulate the expression of several genes in the recently described CSC-enriched Yes-Associated Protein (YAP) signature. ATRA-inducible genes of the YAP signature were associated with good prognosis, whereas ATRA-repressible genes were associated with poor prognosis, depending on the subtype of breast cancer. ATRA reduced mammosphere forming ability of breast cancer cells including acquired anti-estrogen resistant cells, which correlated with ATRA-mediated reduction of the critical stemness-associated gene SOX2 and induction of SOX21, which functions as a differentiation factor depending on the cell type. CD44+/CD24- breast cancer cell line with K-ras mutation was resistant to ATRA, which was reversed by MAP kinase inhibitors. From these results, we propose ATRA as a potential therapy for specific subtypes of breast cancers enriched for CSCs. A biomarker-driven patient selection may help to identify patients who may benefit from ATRA therapy.

Citation Format: Poornima Bhat-Nakshatri, Chirayu Goswami, Sunil Badve, George W. Sledge, Harikrishna Nakshatri. Connectivity-map analyses identify FDA-approved drugs targeting breast cancer stem 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 230. doi:10.1158/1538-7445.AM2013-230
Abstract 2306: MicroRNA 199a*: a potent suppressor of tumor metastasis and angiogenesis.
Authors: Ghosh Alip , Ghosh Amit , Dasgupta Debanjali , Roychowdhury Shrabasti , Roychowdhury Shrabasti , Datta Simanti , Chowdhury Abhijit , Banerjee Soma
Published: August 2014

The growth of a solid tumor depends on diffusion of nutrients from the tumor microenvironment through vascular system. Angiogenesis, the new blood vessels formation is the primary route by which tumor cells get nutrients as well as exit from primary site and enter into the blood circulation. The proliferation and migration of vascular endothelial cells are triggered by angiogenic growth factors secreted by tumor cells, such as VEGF, TGF-β etc. Recent evidences showed HGF also plays an important role in angiogenesis by enhancing the proliferative activity and intracellular signaling. HGF/its receptor, c-MET and VEGF/VEGFR1 are two independent pathways for angiogenesis and metastasis. Thus detection of a molecule that hits both pathways could be a potent therapeutic agent that could facilitate the inhibition of improved barrier function. Recently mir199a* has been shown to target both CD44 and cMET molecule to block the metastasis in cancer. The present study aim to show that mir199a* also target HGF, the key modulator of the HGF/c-Met signaling cascade in epithelial cells to shut off the mesenchymal transition and VEGFR1 to prevent angiogenesis more effectively.

Luciferase 3’ UTR assay was performed to investigate HGF as a target of mir-199a*. HGF mRNA and protein expression was determined by real time RT-PCR, Western blot analysis in liver stellate cell line LX2 transfected with mir-199a* and HGF shRNA. The cultured media of mir-199a* transfected LX2 cells was used to verify the effect of mir-199a* on HGF induced migration and invasion ability on metastatic cell lines using Boyden chamber assay with or without matrigel coated membrane and angiogenesis by tube formation assay.

The mir-199a* binding site was detected by pictar, target scan analysis of the 3’UTR of HGF and VEGFRs. The binding of mir-199a* to the 3′UTR of HGF and VEGFR1 were confirmed by luciferase assay, mutagenesis and western blotting. Down regulation of HGF protein expression in HGF producing hepatic stellate cell, LX2 was observed by mir-199a*. Addition of LX2 conditioned media transfected with mir199a* to metastatic cell reduces the migration and invasion and also block angiogenesis in endothelial cells.

As Mir-199a* could target HGF/cMET, CD44-cMET and VEGF/VEGFR1 signaling cascade, it could be used as a potential therapeutic target of HCC.

Citation Format: Alip Ghosh, Amit Ghosh, Debanjali Dasgupta, Shrabasti Roychowdhury, Shrabasti Roychowdhury, Simanti Datta, Abhijit Chowdhury, Soma Banerjee. MicroRNA 199a*: a potent suppressor of tumor metastasis and angiogenesis. [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 2306. doi:10.1158/1538-7445.AM2013-2306
Abstract 232: Cancer stem cells and cisplatin.
Authors: Modur Vishnu
Published: August 2014

Introduction:

Cisplatin is an important chemotherapeutic agent, which has resulted in higher overall response rates when combined with 5-FU or taxane combinations in treating squamous cellular carcinoma of the head and neck. However, HNSCC tumor response rates to cisplatin range from 20-30%, a major clinical hurdle today. To address this issue thoroughly, we investigated the resistance to cisplatin in HNSCC under the purview of the cancer stem cell hypothesis, using the widely studied CSC marker combinations of, CD44+ 24- and ALDH-Hi. In order to get a broader perspective on this issue, we worked on multiple HNSCC tumor cell lines with varying degrees of cisplatin resistance, arising from tumors of the pharynx, larynx, tongue, tonsils, alveolus, hypopharynx and supraglottis.

Methods:

Seven HNSCC cell lines were analyzed for their CSC percentage through FACS, utilizing CD44+ 24- and ALDH-Hi marker systems. Cisplatin IC-50 for each of these seven cell lines was then determined by plotting percent viability vs. drug concentration curves. The remaining viable cells after cisplatin treatment were then analyzed through FACS for their CD44+ 24- and ALDH-Hi percentages in order to compare with the CSC percentage in the untreated HNSCC lines.

Results:

There was a wide variation in the percentages of CD44+24- and ALDH-Hi cells across these seven HNSCC lines. An inverse correlation between the initial percentages of CD44+24- and ALDH-Hi cells and the corresponding cisplatin IC-50 values was seen. HNSCC cell lines that had lower IC-50 values for cisplatin displayed greater cell viability at higher concentrations of cisplatin. This was evident by a long tail in the sigmoidal dose response curve. The number of viable cells at the end of cisplatin treatment had a linear relationship with the overall percentage of CSCs in the control untreated cell lines.

Conclusion:

Our results suggest that the resistance offered by the CD44+24- and ALDH1-Hi cells is apparent only once the initial 50% loss of cells occurs. This in vitro biphasic decline in percent viable cells clearly indicates that HNSCC cisplatin resistance is a twofold problem. The bulk of non-cancer stem cells within a tumor can acquire resistance to cisplatin via multiple molecular pathways, but this is only a front line cover of resistance to the resistance offered by the cancer stem cells.

Citation Format: Vishnu Modur. Cancer stem cells and cisplatin. [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 232. doi:10.1158/1538-7445.AM2013-232