bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5425: Epidermal respiratory uncoupling blocks skin carcinogenesis by inhibiting Akt.
Authors: Nowinski Sara M. , Rundhaug Joyce E. , Rho Okkyung , Lago Cory U. , Fischer Susan M. , DiGiovanni John , Mills Edward M.
Published: August 2014

Metabolic reprogramming, characterized by increased glycolysis and decreased oxygen consumption, is now considered a hallmark of malignancy. Mitochondrial uncoupling proteins oppose this phenotype by driving futile mitochondrial respiration that is uncoupled from ATP synthesis, and mice expressing a keratin 5 - uncoupling protein 3 (K5-UCP3) transgene in the epidermis are completely protected from the formation of chemically induced skin carcinomas. We crossed these mice with Tg.AC mice that express a cutaneous oncogenic v-Ha-Ras transgene, bypassing tumor initiation. Bi-transgenic Tg.AC/K5-UCP3 mice were still remarkably resistant to 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced tumorigenesis, indicating that UCP3 likely inhibits tumor promotion. Accordingly, K5-UCP3 mice exhibited striking resistance to epidermal proliferation induced by TPA as measured using a bromodeoxyuridine (BrdU) based labeling assay. In agreement with these data, K5-UCP3 epidermis failed to show activation of Akt (Ser473 & Thr308 phosphorylation) and phosphorylation of downstream signaling molecules in response to TPA treatment. This effect corresponded to decreased expression of cyclins D1 and A, and increased expression of the cell cycle inhibitory proteins p21 and p27. Decreased Akt activation was also observed in vitro in response to epidermal growth factor (EGF) treatment of serum starved, isolated K5-UCP3 primary keratinocytes. To test the notion that the lack of Akt signaling was responsible for the lack of TPA induced proliferation and tumorigenesis, we bred bi-transgenic K5-UCP3/K5-Akt mice, and found that over-expression of wild type Akt recovered proliferation following TPA treatment. Future experiments will elucidate the mechanism underlying UCP3-induced inhibition of Akt activation and signaling.

Citation Format: Sara M. Nowinski, Joyce E. Rundhaug, Okkyung Rho, Cory U. Lago, Susan M. Fischer, John DiGiovanni, Edward M. Mills. Epidermal respiratory uncoupling blocks skin carcinogenesis by inhibiting Akt. [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 5425. doi:10.1158/1538-7445.AM2013-5425
Abstract 5435: Increased tumor susceptibility to low glucose under hypoxia is increased by BRAF(V600E) mutation: correlation with persistent phosphoP90RSK activation.
Authors: Rieber Manuel , Chavez-Perez Valery , Strasberg-Rieber Mary
Published: August 2014

BACKGROUND.- Oncogenic BRAF(V600E) mutations, found in >50 % of melanomas, promote melanoma cell proliferation by constitutively activating mitogen-activated Mek1/2 kinases and extracellular signal-regulated kinases (Erk1/2). This pathway can further activate downstream the p90- ribosomal S6 kinase (p90RSK). OBJECTIVES.- Since BRAF mutations increase melanoma progression which could lead to metabolic stress when oxygen, glucose, and other nutrients are limiting within poorly vascularized regions, we investigated whether hypoxia and metabolic stress can differentially influence the survival of human melanoma cells harbouring either wt or oncogenic BRAF. EXPERIMENTAL. human melanoma cells with unequal BRAF status were exposed to glucose and/or glutamine limitations under normoxia or hypoxia. Relative metabolic activity was fluorometrically quantitated by the cellular conversion of resazurin to resorufin. Differential cell survival was assesed by crystal violet staining. Mechanisms influencing the response to metabolic stress were studied by specific immune blotting. RESULTS. the metabolic activity and survival of melanoma cells harbouring oncogenic BRAF was preferentially decreased under hypoxia at sub-physiological glucose, compared to wt BRAF melanoma cells. Addition of the anti-oxidant N-acetylcysteine or pyruvate enhanced the metabolic activity and survival of wt BRAF cells, but these were much less effective when added to BRAF (V600E) mutant cells, implying a greater oxidative stress induced by low oxygen and glucose in them. Susceptibility to metabolic stress under hypoxia and low glucose in BRAF (V600E) mutant cells, correlated with persistent (p90RSK) activation but lower GLUT-1 and MnSOD expression. In contrast, wt BRAF melanoma cells resistant to metabolic stress reciprocally showed higher GLUT-1 and MnSOD expression, but negligible p90RSK activation. CONCLUSION.- Although mutations in BRAF can drive acquired resistance to MEK1/2 inhibitors, our results suggest that restricting glucose consumption in hypoxic melanomas harbouring mutant BRAF with persistent phosphorylation of the p90-ribosomal S6 kinase, may contribute to their preferential killing.

Citation Format: Manuel Rieber, Valery Chavez-Perez, Mary Strasberg-Rieber. Increased tumor susceptibility to low glucose under hypoxia is increased by BRAF(V600E) mutation: correlation with persistent phosphoP90RSK activation. [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 5435. doi:10.1158/1538-7445.AM2013-5435
Abstract 5471: Notch-antibody drug conjugates have a different mechanism of action than Notch signaling inhibitors and induce tumor regression.
Authors: Geles Kenneth G. , Gao Yijie , Sridharan Latha , Giannakou Andreas , Yamin Ting-Ting , Golas Jonathan , Charati Manoj , Khandke Kiran , Lucas Judy , Maderna Andreas , O'Donnell Christopher J. , Tchistiakova Lioudmila , Gerber Hans-Peter , Sapra Puja
Published: August 2014

There are four different Notch receptors in mammalian cells that have overlapping patterns of expression in embryonic and adult tissues, but fulfill non-redundant roles during hematopoietic stem cell specification, T cell development, intestinal crypt cell specification and vascular development. Notch receptors are over-expressed or amplified in certain human tumors and regulate cell proliferation, differentiation, and survival through an intracellular domain that functions as a transcriptional activator. Several strategies are in development to block Notch signaling for therapeutic purposes in cancer, including gamma-secretase inhibitors that block all Notch signaling and antibody-based targeting of individual receptors. However, blocking pathway activation with inhibitory antibodies has proven to be less efficacious than originally anticipated. Therefore, we have targeted Notch with antibody-drug conjugates (ADCs) that combine the specificity of high affinity anti-Notch antibodies with the cytotoxicity of microtubule inhibitors. These ADCs enhance efficacy and also allow targeting in tumors that overexpress Notch but are not driven by its signaling. Notch antibodies were conjugated though cleavable and non-cleavable linkers to novel tubulin inhibitor-based payloads. Notch-ADCs inhibited the in vitro growth of lung, breast and ovarian cancer cell lines in the low ng/ml range and in vivo regressed the growth of established human tumor xenografts. Our data demonstrate that Notch-ADCs are more potent than small molecule inhibitors and unconjugated antibodies, and induce sustained tumor regression in pre-clinical models.

Citation Format: Kenneth G. Geles, Yijie Gao, Latha Sridharan, Andreas Giannakou, Ting-Ting Yamin, Jonathan Golas, Manoj Charati, Kiran Khandke, Judy Lucas, Andreas Maderna, Christopher J. O'Donnell, Lioudmila Tchistiakova, Hans-Peter Gerber, Puja Sapra. Notch-antibody drug conjugates have a different mechanism of action than Notch signaling inhibitors and induce tumor regression. [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 5471. doi:10.1158/1538-7445.AM2013-5471
Abstract 5509: Natural occurring leelamine inhibits melanoma development by targeting multiple signaling pathways.
Authors: Gowda Raghavendra , Madhunapantula SubbaRao V. , Kuzu Omer F. , Sharma Arati , Gavin Robertson P.
Published: August 2014

Melanoma remains a highly drug resistant tumor type. Melanoma cells rapidly progress resistance to drugs targeting single proteins by circumventing the point of inhibition. To overcome this problem, one approach has been to develop drugs targeting multiple proteins or pathways deregulated in the growth of the disease, such as the PI3 kinase, MAP kinase, and STAT3 signaling cascades. Agents simultaneously inhibiting several key pathways aiding melanoma development would be a first in class type of new drug for treating melanoma. To identify a drug that might target the PI3K, MAPK and STAT3 cascades, a natural product library was screened identifying leelamine as a potential inhibitor. Leelamine had the greatest inhibitory efficacy selectively killed melanoma cells by decreasing levels of cellular proliferation and increasing apoptosis rates. Leelamine inhibited the growth of preexisting xenografted tumors by ∼60%, by targeting PI3K, MAPK and STAT3 pathways without affecting animal body weight or blood markers of major organ function. The most prominent tumor inhibitory mechanisms of actions of leelamine occurred through by decreasing of Akt, Erk and Stat3 activity, which reduced cellular proliferation and consequently decreased tumor vascularization as well as increasing apoptosis. Collectively, these discoveries demonstrate the development of a naturally occurring pharmacological agent that targets multiple pathways and thereby more effectively treat melanoma.

Citation Format: Raghavendra Gowda, SubbaRao V. Madhunapantula, Omer F. Kuzu, Arati Sharma, Robertson P. Gavin. Natural occurring leelamine inhibits melanoma development by targeting multiple signaling pathways. [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 5509. doi:10.1158/1538-7445.AM2013-5509
Abstract 5512: Mechanisms of inhibition of cancer cell growth and metastasis by novel small molecule compounds.
Authors: Ashour Abdelkader E. , Cheryan Vino T. , Jamal Shazia , Alafeefy Ahmed M. , Zoheir Khairy M. , Abdallah Adel R. , Rishi Arun K.
Published: August 2014

The molecular complexity of cancers and therapy-associated side effects often limit effectiveness of many therapies, and warrant development of new agents for specific molecular targets while minimizing the off-target effects. Thus, our goal is to develop novel, safer and effective anti-cancer therapies on the basis of cell growth and survival inhibiting functions of an apoptosis regulatory protein, termed CARP-1. CARP-1 was identified by us as a novel target of select chemotherapy (adriamycin, etoposide, or Iressa)-dependent human breast cancer cell growth suppression. By conducting the high-throughput screening of chemical libraries, we identified a number of novel, small molecule compounds called CARP-1 Functional Mimetics (CFMs). MTT assay results showed that CFMs, as well as quinazoline analogues of CFMs, inhibited growth of a variety of cancer cells, including the drug-resistant breast cancer cell line MDA-MB 468, without affecting growth of immortalized mammary epithelial cells. Results of MTT assay indicated also that CFM-4 (1(2-chlorobenzyl)-5′-phenyl-3′H-spiro[indoline-3,2′-[1,3,4]thiadiazol]-2-one) was the most potent one. CFMs suppressed cancer cell growth in part by stimulating CARP-1 levels and promoting cell cycle arrest and apoptosis. RT-PCR assay results revealed that CFM-4 induced the extrinsic pathway of apoptosis via upregulation of TRAIL receptors, caspase-8 and caspase-3 which can then cleave multiple cellular proteins, leading to cancer cell death. Cancer-related deaths are often due to spread of cancers to other organ sites (metastasis) that involves invasion and movement of cancer cells through the nearby normal tissue and the lymph and blood vessels to reach other tissues to form secondary tumors. Here we also demonstrate that CFMs impact biological properties of cancer cells by inhibiting their movement and invasion through the membranes coated with the extra-cellular matrix. CFMs also inhibited abilities of cancer cells to grow as colonies in soft-agar. These proof-of-concept studies suggest that CFMs could induce cancer cell apoptosis and effectively inhibit migration and tumor-forming abilities of cancer cells. Thus CFMs or their analogues have potential as anti-cancer agents to target cancer cell proliferation, survival, migration, invasion and metastasis, and provide a strong basis for their development as anti-cancer agents.

Citation Format: Abdelkader E. Ashour, Vino T. Cheryan, Shazia Jamal, Ahmed M. Alafeefy, Khairy M. Zoheir, Adel R. Abdallah, Arun K. Rishi. Mechanisms of inhibition of cancer cell growth and metastasis by novel small molecule compounds. [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 5512. doi:10.1158/1538-7445.AM2013-5512
Abstract 5513: Discovery of an orally active small-molecule irreversible inhibitor of protein disulfide isomerase (PDI) for ovarian cancer treatment.
Authors: Xu Shili , Butkevich Alexey N. , Yamada Roppei , Zhou Yu , Debnath Bikash , Duncan Roger , Zandi Ebrahim , Petasis Nicos A. , Neamati Nouri
Published: August 2014

Protein disulfide isomerase (PDI), an endoplasmic reticulum (ER) chaperone protein, catalyzes disulfide bond breakage, formation and rearrangement. The effect of PDI inhibition on ovarian cancer progression is not yet clear, and there is a need for potent, selective and safe small-molecule inhibitors of PDI. Here, we report a new class of novel propynoic acid carbamoyl methyl amides (PACMAs) that are active against a panel of human ovarian cancer cell lines. Using fluorescent derivatives, 2D gel electrophoresis, and mass spectrometry, we established that PACMA 31, one of the most active analogues, acts as an irreversible small-molecule inhibitor of PDI, forming a covalent bond with PDI's active site cysteines. We also demonstrated that PDI activity is essential for the survival and proliferation of human ovarian cancer cells. In vivo, PACMA 31 showed tumor targeting ability and significantly suppressed ovarian tumor growth without causing toxicity to normal tissues. These irreversible small-molecule PDI inhibitors represent a new approach for the development of targeted anticancer agents for ovarian cancer therapy, and can also serve as useful probes for investigating the biology of PDI-implicated pathways.

Citation Format: Shili Xu, Alexey N. Butkevich, Roppei Yamada, Yu Zhou, Bikash Debnath, Roger Duncan, Ebrahim Zandi, Nicos A. Petasis, Nouri Neamati. Discovery of an orally active small-molecule irreversible inhibitor of protein disulfide isomerase (PDI) for ovarian cancer treatment. [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 5513. doi:10.1158/1538-7445.AM2013-5513
Abstract 5514: Antitumor activity of (R,R’)-4′-methoxy-1-naphthylfenoterol in a rat C6 glioma xenograft model in the mouse.
Authors: Paul Rajib K. , Ramamoorthy Anuradha , Zhang Yongqing , Becker Kevin G. , Paris Arnaud , Cloix Jean-Francois , Bernier Michel , Wainer Irving W.
Published: August 2014

(R,R’)-4’-methoxy-1-naphthylfenoterol (MNF) inhibits in vitro proliferation of several types of cancer cell lines. In this study, the in vivo antitumor effects of MNF were evaluated using rat C6 glioma cells implanted subcutaneously into the lower flank of 5 week-old NMRI/Nude female Swiss mice (0.5 x 10(6) cells, 100μl). Three days after the inoculation, the mice were subjected to intraperitoneal injections of saline or MNF (10μl/g, 2mg/kg of body weight) for 5 days per week for 2 weeks. Tumor volumes were measured everyday using slide calipers. Significant reductions in mean tumor volumes were observed in mice receiving MNF when compared with the saline-treated group (p < 0.001, n=17-19). At the end of the study, animals were sacrificed and tumors were collected for cDNA microarray, quantitative PCR and immunoblot analyses. Comparison of cDNA microarray analysis of tumors from mice treated with MNF vs. saline demonstrated a significant decrease in expression of genes involved in cellular proliferation, including mitotic checkpoint kinase MAD3L (BUB1B), cyclin-dependent kinase inhibitor 3 (CDKN3) and cyclin A2 (CCNA2), as well as molecular markers for glioblastoma, such as oligodendrocyte transcription factor 1 (OLIG1) and SRY-box 4 (SOX4). Target gene validation is currently in progress. Our results show that MNF reduces growth of flank xenografted C6 glioma tumors and expression of genes related to tumor proliferation and suggest that this compound may be useful in cancer treatment. This work was supported in part by the Intramural Research Program of the NIA/ NIH.

Citation Format: Rajib K. Paul, Anuradha Ramamoorthy, Yongqing Zhang, Kevin G. Becker, Arnaud Paris, Jean-Francois Cloix, Michel Bernier, Irving W. Wainer. Antitumor activity of (R,R’)-4′-methoxy-1-naphthylfenoterol in a rat C6 glioma xenograft model in the mouse. [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 5514. doi:10.1158/1538-7445.AM2013-5514
Abstract 5518: A novel 1,3-bis(3,5-dichlorophenyl) urea compound ‘COH-SR4’ for targeting melanoma.
Authors: Dalasanur Nagaprashantha Lokesh Prasad Gowda , Figarola James , Singhal Jyotsana , Horne David , Jove Richard , Rahbar Samuel , Awasthi Sanjay , Singhal Sharad S
Published: August 2014

The current clinical modalities in the management of malignant melanomas are met with limited response. The molecular events that regulate oncogenic transformation, invasive progression and resistance to therapy in melanomas are determined by an interplay between various complex signaling networks. In the context of evident demographic risk, limited response to current clinical interventions and molecular complexity of melanomas, novel candidate molecules capable of effectively inducing tumor inhibition along with targeting critical nodes of melanoma signaling assume translational significance. In this regard, we investigated the anti-cancer effects of a novel dichlorophenyl urea compound called SR4 in melanoma. The SR4 treatment decreased the survival and inhibited the clonogenic potential along with inducing apoptosis in vitro cultures of melanoma. SR4 treatments lead to inhibition of GST activity along with inducing G2/M phase cell cycle arrest. Oral administration of 4 mg/kg b.w. SR4 leads to effective inhibition of tumor burdens in both syngeneic and nude mouse models of melanoma. The SR4 treatment was well tolerated and no overt toxicity was observed. The histopathological examination of resected tumor sections reflected decreased blood vessels, decrease in the levels of angiogenesis marker, CD31, and proliferation marker, Ki67, along with an increase in pAMPK levels. Western blot analyses of resected tumor lysates revealed increased PARP cleavage, Bim, pAMPK along with decreased pAkt, vimentin, fibronectin, CDK4 and cyclin B1. Thus, SR4 represents a highly effective candidate for the further development of single agent and combinatorial therapies to effectively target aggressive and clinically resistant melanomas (Supported by NIH grant CA 77495).

Citation Format: Lokesh Prasad Gowda Dalasanur Nagaprashantha, James Figarola, Jyotsana Singhal, David Horne, Richard Jove, Samuel Rahbar, Sanjay Awasthi, Sharad S Singhal. A novel 1,3-bis(3,5-dichlorophenyl) urea compound ‘COH-SR4’ for targeting melanoma. [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 5518. doi:10.1158/1538-7445.AM2013-5518
Abstract 5532: A pro-fluorescent membrane integrity dye for the real-time assessment of cytotoxicity.
Authors: Somberg Richard L. , Niles Andrew , Worzella Tracy , Lazar Dan
Published: August 2014

Cell-based assay models are now universally employed as a routine means of establishing and ranking on- and off-target toxicities. It is well appreciated, however, that individual cytotoxic phenotypes are shaped not only by compound dosage, but by the length of compound-cell exposure. In addition to compound-specific attributes, inherent cellular factors such as biotransformation capacity, cell cycle susceptibility, and receptor expression often dictate the kinetics of cytotoxicity. Unfortunately, the majority of conventional cytotoxicity assays are performed at a terminal endpoint (48-72h) which at worst can underestimate cytotoxicity due to biomarker degradation, or at best, fail to reveal important features of the cytotoxic event which may be important for establishing mechanism-of-action. We have developed a pro-fluorescent, cell-impermeant probe which can be applied to cells at the time of dosing to report changes in membrane integrity as they occur in real-time. The dye enters cells with impaired membrane integrity, greatly enhancing fluorescence which is proportional to non-viable cell number. The dye can be used in traditional plate based formats using standard fluorometry or with image based readers (Essen Bioscience Incucyte™) to establish the dose-dependent kinetics of cytotoxicity. Here we describe the dye's utility using mechanistically distinct cytotoxins with both cancer cell lines (on-target) and terminally differentiated cells (off-target) and correlate this dead cell fluorescence activity to other cytotoxicity biomarkers by same-well multiplexed methods.

Citation Format: Richard L. Somberg, Andrew Niles, Tracy Worzella, Dan Lazar. A pro-fluorescent membrane integrity dye for the real-time assessment of cytotoxicity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5532. doi:10.1158/1538-7445.AM2013-5532
Abstract 5535: Development of peptidomimetic inhibitors of the ERG transcription factor in prostate cancer.
Authors: Wang Xiaoju
Published: August 2014

Transcription factors play a key role in the development of a number of cancers, and therapeutically targeting them has remained a challenge. In prostate cancer, the ETS transcription factor ERG is recurrently rearranged and likely plays a critical role in prostate oncogenesis. Here we identified a consensus peptide from a phage library which interacts specifically with the DNA binding domain of ERG. The interactive interface was mapped to 9-residues in the 3rd α-helix of the ETS domain, which is critical for ERG activity as a transcription factor. The peptides were found to efficiently disrupt ERG-mediated protein-protein interactions, transcription, DNA damage, and cell invasion. Furthermore, a retroinverso peptidomimetic suppressed tumor growth, intravasation, and metastasis in vivo. Taken together, our results suggest that transcription factors have specific residues that are important for protein-protein interactions, that may be amenable to therapeutic targeting.

Citation Format: Xiaoju Wang. Development of peptidomimetic inhibitors of the ERG transcription factor in prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5535. doi:10.1158/1538-7445.AM2013-5535

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 5593: A cancer-associated hyperphosphorylation site on topoisomerase I serine 506 as a biomarker for camptothecin sensitivity.
Authors: Bandyopadhyay Keya , Li Pingchuan , Gjerset Ruth A.
Published: August 2014

Topoisomerase I (topo I) plays a central role in cancer as the target of a potent class of chemotherapeutic drugs derived from the plant alkaloid camptothecin that includes irinotecan and topotecan. We have identified a novel cancer-specific phosphorylation site at serine 506 in topo I (PS506), targeted by protein kinase CK2, that identifies cancer cells with elevated CK2, hyperphosphorylated topo I, increased topo I activity, and increased sensitivity to camptothecin. CK2 treatment of recombinant topo I in vitro also induces topo I hyperphosphorylation and expression of the PS506 epitope, and results in increased binding of topo I to supercoiled plasmid DNA. The hyperphosphorylated, PS506-form of topo I is more effective than the basal phosphorylated enzyme at relaxing plasmid supercoils but has a similar catalytic rate as the basal phosphorylated enzyme once bound to DNA. The PS506 epitope is not detected in normal cells or cancer cell lines with lower levels of CK2 activity. By experimentally manipulating CK2 activity in cancer cell lines, we have demonstrated a cause and effect relationship between CK2 activity, PS506 expression, camptothecin-induced cellular DNA damage, and cellular camptothecin sensitivity. The PS506 epitope is therefore an indicator of dysregulated, hyperphosphorylated topo I in cancer cells, and holds promise as a novel diagnostic biomarker that predicts tumor responsiveness to widely used topo I-targeted therapies.

Citation Format: Keya Bandyopadhyay, Pingchuan Li, Ruth A. Gjerset. A cancer-associated hyperphosphorylation site on topoisomerase I serine 506 as a biomarker for camptothecin sensitivity. [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 5593. doi:10.1158/1538-7445.AM2013-5593
Abstract 5594: JNK1 and MERTK are markers of MEK inhibitor resistance and new targets for therapy.
Authors: Beech Jaymes , Kelly Kim
Published: August 2014

Pancreatic ductal adenocarcinoma (PDAC) is one of the most devastating human cancers, having a median survival of 6 months and a 5-year survival rate of 6%. It is the fourth leading cause of cancer deaths due to its resistance to conventional radiotherapy and chemotherapy and a lack of early detection. Alternatively, small molecule inhibitors have been emerging as a means of transitioning away from non-selective treatments towards a more personalized approach.

Pancreatic cancer, having mutant K-RAS and TP53 in upwards of 90% of cases, appears to be a great candidate for molecular therapy due to this high genetic homogeneity among patients. TP53 is an inactivation mutation and not a candidate for inhibition. K-RAS is an over-activating mutation but unfortunately cannot currently be treated by small molecule inhibitors. However, its downstream pathways, which signal both growth and survival, can. The most predominant of these is the MAP kinase pathway. Several small molecules have been developed to target members of this pathway such as MEK 1/2 including U0126, PD0325901 and AZD6244. Often, however, these therapies are still ineffective and it would be immensely beneficial to be able to predict when MEK inhibition will be effective or should conversely be avoided.

To discover such markers, we tested a panel of 18 pancreatic cancer cell lines for sensitivity to the MEK inhibitor AZD6244. After classifying each line's sensitivity, microarray and qPCR analysis found significantly different genes between the sensitive and resistant groups. Most notable were MERTK and MAPK8, which both were upregulated in resistant lines and showed some redundancy in the studies. These genes code for the Tyrosine-protein kinase Mer (MERTK) and c-Jun N-terminal kinase (JNK1) proteins respectively. Correlation studies showed both gene and protein expression levels to track with resistance to the small molecule inhibitor. To test whether these genes and the proteins they code for could serve as potential new therapy targets, shRNA studies were performed. Studies yielded a noticeable decrease in proliferation in both MAPK8 and MERTK knockdown, even being lethal in one case. While the results support the importance of these proteins for tumorgenecity, knockdown did not alter the cell lines’ sensitivity to MEK inhibition. Our findings suggest the use of MERTK and JNK1 as markers of MEK inhibition resistance and propose new target for future therapies.

Citation Format: Jaymes Beech, Kim Kelly. JNK1 and MERTK are markers of MEK inhibitor resistance and new targets for therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5594. doi:10.1158/1538-7445.AM2013-5594
Abstract 5600: Destruction of prostate cancer cell xenografts by FSH-Lytic peptide conjugates.
Authors: Aggarwal Sita , Gauthier Ted , Alila Hector , Leuschner Carola , Karki Namrata , Solipuram Rajasree , Hansel William
Published: August 2014

Background: In previous studies (Hansel, et al., Mol. and Cell. Endocrinol. 269:26-33, 2007),we showed that conjugates of membrane destroying lytic peptides with either LHRH or with a 15-amino acid segment of the β chain of human chorionic gonadotropin (hCG) target and destroy human prostate, breast and ovarian cancer cells in tumor bearing nude mice. Recently (Radu et al., N. England J. Med. 363:1621-1630, 2012) reported that endothelial cells in blood vessels supplying cancers express follicle-stimulating hormone (FSH) receptors.

Objectives:The objectives of this study were to synthesize a bioconjugate of a lyticpeptide (Phor18) to each of three segments of the β chain of FSH that are known to bind to the FSH receptor, and test these conjugates (FSH90-95-Phor18, FSH81-95-Phor18 and FSH33-53-Phor18) for their ability to target and lyse prostate cancer cells in vitro and in vivo.

Results: In in vivo experiments, administration of FSH90-95-Phor18 and FSH81-95-Phor18 significantly (p < 0.05) inhibited prostate cancer cell (PC-3) growth, as measured by tumor volumes and tumor weights at necropsy. FSH33-53-Phor18 also caused a significant reduction in tumor volume at necropsy at a dose of 1 mg/kg, but the reduction in tumor weight was not statistically significant (p > 0.05). The average tumor volume was maintained at significantly lower levels in the mice treated with FSH90-95-Phor18 (p=0.027), FSH81-95-Phor18 (p=0.029) when compared with vehicle and free peptide treatment groups during the study (p < 0.05). Immunohistochemical analyses for FSHR were performed on the tumor samples. FSHR-positive endothelial cells were found in many vessels supplying the tumors of control mice, but few FSHR-positive endothelial cells were present in tumors of mice treated with FSH90-95-Phor18, FSH81-95-Phor 18 or FSH33-53-Phor 18. FSH81-95-Phor 18 was the most effective of the three conjugates tested in destroying the FSHR-bearing endothelial cells and in inhibiting tumor growth of the PC-3 xenografts in nude mice.

Conclusion: These data show that Phor18 conjugates of FSH β chain segments that bind to FSHR expressed by the endothelial cells of the blood vessels supplying the tumors are capable of inhibiting prostate cancer cell tumor growth by inhibiting angiogenesis. These FSH β chain-lytic peptide conjugates may be useful in treating prostate and other FSHR expressing cancers.

Citation Format: Sita Aggarwal, Ted Gauthier, Hector Alila, Carola Leuschner, Namrata Karki, Rajasree Solipuram, William Hansel. Destruction of prostate cancer cell xenografts by FSH-Lytic peptide conjugates. [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 5600. doi:10.1158/1538-7445.AM2013-5600
Abstract 5638: Preclinical evidence supporting cotargeting insulin-like growth factor receptor (IGF-1R) and Src in non-small cell lung cancer.
Authors: Kim Jin-Soo , Min Hye-Young , Yun Hye Jeong , Jang Hyun-Ji , Kim Edward S. , Liu Diane , Lee J. Jack , Behrens Carmen , Lippman Scott M. , Hong Waun Ki , Wistuba Ignacio I. , Lee Ho-Young
Published: August 2014

The antitumor efficacy of IGF-1R targeting agents (TKIs or antibodies) has been evaluated in recent clinical trials, but the effect has been found to be marginal and limited. Since it has been suggested that IGF-1R and Src signaling pathways cooperate with each other in the proliferation and survival of cancer cells, we investigated the role of Src in the resistance to the IGF-1R TKI and further suggested the rationale of co-targeting of IGF-1R and Src in the treatment of NSCLC. We examined Src-mediated regulation of IGF-1R activation and assessed the effect of the combined treatment with an IGF-1R TKI and a Src inhibitor on the proliferation, survival, and signaling changes in human NSCLC cells and NSCLC xenografts in nude mice. In addition, expressions of the IGF-1R signaling axis were evaluated in tissue microarrays of NSCLC from two independent databases (N=352, and 353, respectively) and correlated with clinicopathologic characteristics and patient survival. We found co-activation of IGF-1R and Src in various NSCLC cells and modulation of IGF-1R signaling by overexpression or knockdown of Src. Combined blockade of IGF-1R and Src resulted in the suppression of cell proliferation, induction of apoptosis, inhibition of signaling responsible for cell survival and proliferation, and tumor growth in vivo. In human tissue samples, p-IGF-1R/IR, IGF-1R, and pSrc expressions were significantly associated with squamous cell carcinoma (SCC) in both TMAs. pIGF-1R/IR and pSrc had strong positive correlation with each other (P<.0001 and P<.0001, respectively). The expression of pSrc serves as a predictor of poor survival in these patients by multivariate analysis in patients with adenocarcinoma. Our data suggest that Src may play an important role in the resistance of a small molecule inhibitor targeting IGF-1R, and IGF-1R and Src are activated in NSCLC patients and thus should be investigated as therapeutic targets in NSCLC patients. Further studies would be necessary to examine the efficacy of combined treatment targeting IGF-1R and Src in NSCLC patients.

Citation Format: Jin-Soo Kim, Hye-Young Min, Hye Jeong Yun, Hyun-Ji Jang, Edward S. Kim, Diane Liu, J. Jack Lee, Carmen Behrens, Scott M. Lippman, Waun Ki Hong, Ignacio I. Wistuba, Ho-Young Lee. Preclinical evidence supporting cotargeting insulin-like growth factor receptor (IGF-1R) and Src in non-small cell lung 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 5638. doi:10.1158/1538-7445.AM2013-5638
Abstract 5667: Cytotoxicity of two-component drug delivery systems targeting HER2(+) cancer cells.
Authors: Hapuarachchige Sudath , Kato Yoshinori , Artemov Dmitri
Published: August 2014

The HER2 (ErbB-2) is a class of epidermal growth factor receptors, which is overexpressed in 20-30% breast cancer cases that consequently show poor prognosis, cancer growth, and progression. Trastuzumab is a humanized, monoclonal antibody widely used as a first line therapeutic against HER2(+) breast cancers. However, recent clinical observations reveal that approximately half of patients with HER2(+) disease do not benefit from trastuzumab or have disease that becomes refractory to it. The combination therapy based on antibody-drug conjugates is a promising strategy to overcome the immunotherapeutic resistance of tumors. Since single delivery components face the barriers of extravasation from vessels and diffusion to the target site, we have developed a sequential two-component delivery system where components are assembled on the target cell surface and internalized delivering high dose of therapeutics. In this study, trastuzumab was functionalized with PEGylated azide groups and labeled with rhodamine or NIR CF-680 dyes, to obtained T(PEG4-Az)8(Rhod/CF-680)2 as a first delivery component. Albumin (BSA) was functionalized with strain-promoted alkyne (DIBO), substituted with paclitaxel, and labeled with Alexa Fluor 488 or CF-750 to obtained A(DIBO)10(Pac)2(Alexa 488/CF-750)2 as the second delivery component. Substitution of paclitaxel was carried out with sulfo-NHS-paclitaxel which increases the solubility of the drug in reaction medium. While reduced solubility of paclitaxel modified BSA was observed for the substitution of >5 paclitaxel molecules, one to four paclitaxel substitutions on BSA shows no precipitations in PBS. The in vivo cytotoxicity of the two-component delivery system was assessed using HER2(+) overexperssing BT-474 breast cancer cells and a standard WST-8 assay. For in vitro therapeutic study, we used T(PEG4-Az)8(CF-680)2 and A(DIBO)10(Pac)2(CF-750)2 (with non-functionalized trastuzumab as the first component in controls). We observed an increased cytotoxicity of the two-component delivery system with IC50 of 0.101 μM, comparing to IC50 of 0.247 μM for the control.

Citation Format: Sudath Hapuarachchige, Yoshinori Kato, Dmitri Artemov. Cytotoxicity of two-component drug delivery systems targeting HER2(+) 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 5667. doi:10.1158/1538-7445.AM2013-5667
Abstract 579: The interaction of ZSCAN4 with TRF1: effects on regulation of telomere elongation in cancer cells.
Authors: lee kyungwoo
Published: August 2014

Telomeres are repetitive sequences at the ends of chromosomes protected by DNA binding proteins of shelterin complex that form capping structures. One of these proteins, TRF1, plays a role in the regulation of telomere length through its interaction with other proteins in the shelterin complex. High levels of TRF1 expression have been shown to accelerate telomere shortening, whereas dominant-negative inhibition of TRF1 leads to telomere elongation. Recently, the newly identified Zinc finger and SCAN domain containing4 gene (ZSCAN4) plays a key role in genomic stability by regulating telomere elongation and was shown to co-localize with TRF1 foci in mouse embryonic stem cell. Here, we show that ZSCAN4 is a TRF1 association protein in breast cancer cells. We hypothesized that ZSCAN4 would affect levels of TRF1expression and correspondingly control the function of TRF1 through negative regulation and dominant-negative inhibition. Using Co-immunoprecipitation and TNT Pull-down assay, we demonstrate that ZSCAN4 binds with TRF1 in vitro. Localization and visualization of interacting protein-protein interactions in living cells are crucial to elucidate between TRF1 and ZSCAN4. Using a bimolecular fluorescence complementation (BiFC) assay, we show the directly visualize TRF1 and ZSCAN4 interactions in living cells. Using telomere length assays, we demonstrate that ZSCAN4 is an association protein of TRF1 to regulate telomere and affect in the telomere elongation of cancer. The ZSCAN4 mediated cooperative binding of TRF1 to telomeric DNA has important implications for understanding the mechanism of telomere extension.

Citation Format: kyungwoo lee. The interaction of ZSCAN4 with TRF1: effects on regulation of telomere elongation 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 579. doi:10.1158/1538-7445.AM2013-579
Abstract 600: Liposomal C6 ceramide appears to potentiate enhanced chemotoxicity of pancreatic cancer.
Authors: Wanebo Harold J. , Cong Cao , Shu Li , Shrayer David , Wan Yeushung , Bowen Wayne
Published: August 2014

Introduction: Pancreatic adenocarcinoma a highly lethal malignancy (5 yr survival ∼ 5%) is poorly responsive to all chemotherapy, in part related to presence of chemo resistant pathways: prosurvival (AKT/PI3K/mTOR and mutant KRAS. We previously demonstrated reversal of these resistant pathways by C6 Ceramide and now pursue optimum intracellular delivery using selected liposomal preparations of C6 Ceramide.

Methods: C6 Ceramide(C6Cer) in 4 different liposomal vesicle formulations were tested by MTT assay against pancreatic cell lines L3.6, PANC1 and MIA, combined with chemotherapy: Gemcitabine, Paclitaxel and Cetuximab.Liposomal preparations included : (#1)18:1 PC (1,2-dioleoyl-sn-glycero-3-phosphocholine; (#3),18:0 PC (1,2-distearoyl-sn-glycero-3-phosphocholine;(#5) DPGG (1,2-dipalmitoyl-sn-glycero-3-galloyl); and (#7) 18:0 PEG2 PE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N[methoxy(polyethylene glycol)-2000. (#1)18:1 PC- has low transition temp.(T*) and is fluid at body T*(# 3). 18:0 PC-is rigid with high transition (T*) . #5 DPGG- avoids RE System (is fluid at body T*, induces long circulations)#7. 18:0 PEG 2 PE - industry standard (also avoids RE system).Biologic effects of the 4 lipid formulations with/without C6 Ceramide were initially assessed invitro.

Results: Dose response MTT assay demonstrated no distinctive response of C6 combined liposomes vesicles # 1 & 3, but confirmed high activity in preparations #5 & 7.

MTT ResponseFractionControlTaxolGemcitabineCetuximab100%87%70%90%#5(C6)90%42%40%33%# 6 PPS control108%76%36%77%# 7 C6101%31%45%59%# 8 PBS control110%60%58%62%*Control-cell survival 72 hours ∼ 90%-100%(Drug Dose - C6 1 ug/ml, Pac. 1ug/ml,Gem 5ug/ml, Cetux 25ug/ml)

Conclude: Liposomal C6 preparations show high activity in potentiating chemo toxicity by Gemcitabine, Paclitaxel and Cetuximab against Pancreatic Cancer. Confirmatory in vivo studies are in process.

Citation Format: Harold J. Wanebo, Cao Cong, Li Shu, David Shrayer, Yeushung Wan, Wayne Bowen. Liposomal C6 ceramide appears to potentiate enhanced chemotoxicity of pancreatic cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 600. doi:10.1158/1538-7445.AM2013-600
Abstract 604: Styrene compounds induce apoptosis in triple negative breast cancer cell lines.
Authors: Rorie Checo J. , Richardson Shaina L. , Lucas Agape C. , Thomas Phillip A. , Gurley Kashenya M. , Franks Marion A.
Published: August 2014

Triple negative breast cancer (TNBC) is a rare basal-like subtype of breast cancer that is highly aggressive due to its invasiveness and metastatic properties. TNBC is characterized by the loss or low expression of estrogen receptor, progesterone receptor, and HER2 proteins which have been targeted and established as somewhat effective therapy regiments in other breast cancer subtypes including luminal breast cancer. Since triple negative tumors are missing these established drug targets, there has been little advancement in the establishment of drug therapies to specifically target this breast cancer subtype and therefore the need for novel therapeutics in treating TNBC still remains. Previous reports have suggested that PARP inhibitors show promise in potentiating the effectiveness of other known chemotherapeutics. Styrene compounds are volatile organic compounds that have a similar structural composition to some known PARP inhibitors. Our group has developed styrene compounds composed of a 3-nitro group on the A ring and varying aryl groups in the B ring of the styrene, and then tested their biological effects on luminal and TNBC cell lines. In this study, four different styrene compounds were used to treat the luminal breast cancer cell line HCC70 and the triple negative breast cancer cell line HCC1806. The breast cancer cells were exposed to a 10, 100, and 1000 μM titration over a 24hr time period. The apoptotic effect on these cell lines were analyzed and measured via qualitative and quantitative methods. We show here that light microscopy revealed a classic apoptotic dose-response to all of the styrene compounds in the cell lines. We also used the cell viability and MTT assays to reveal a dose-response decrease in cell viability and increase in cell death to the titration of the styrene compounds. This preliminary data show that these compounds have the ability to induce apoptosis in luminal and triple negative breast cancer cell lines. Our preliminary results lead us to believe that these styrenes could represent a new chemotherapeutic treatment for Triple Negative Breast Cancer and potentially other breast cancer subtypes.

Citation Format: Checo J. Rorie, Shaina L. Richardson, Agape C. Lucas, Phillip A. Thomas, Kashenya M. Gurley, Marion A. Franks. Styrene compounds induce apoptosis in triple negative breast cancer cell lines. [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 604. doi:10.1158/1538-7445.AM2013-604
Abstract 609: Novel techniques for identifying apoptosis with pSIVA-IANBD in live and fixed cells.
Authors: Heiden Lisa S. , Krajewski Stan , Quintel Payton D. , Singh Sujay K.
Published: August 2014

Historically, flow cytometry has been the primary technique for identifying individual cells undergoing apoptosis. However, imaging is becoming increasingly important for analyzing cell death and underlying mechanisms in living cell populations. In this study, we developed new techniques for using the polar sensitive pSIVA-IANBD Annexin XII probe to identify apoptosis in mixed populations of cells by multiple imaging of viable primary neuronal cultures over 0-72h. The pSIVA-IANBD probe was added to living cultures at time=0. pSIVA-IAND binds to exposed phosphatidylserine (PS) residues and fluoresces (in the FITC channel) only when bound to cells. Cultures were fixed and permeabilized at the end of their respective time courses and immunostained with MAP2 or NeuN neuronal markers to identify neurons, and counterstained with Hoeschst dye to visualize nuclei.

The results were imaged by an IFL multichannel inverted digital microscope (EVOS; AMG Inc, USA). They showed that bound pSIVA-IANBD was fixation resistant and the single (pSIVA-IANBD), double (pSIVA-IANBD + Hoescht) and as well as the triple (pSIVA-IANBD + Hoescht + antibody) stained cells were readily identifiable in the mixed cell population. They suggested that pSIVA-IANBD enables detection of PS, a hallmark of apoptosis by imaging of viable as well as post-fixed cells. Furthermore, since pSIVA-IANBD is not toxic to cells and does not require a washing step, it can be maintained in cultures with only a single application and no medium change for up to 72h. This allows continuous live monitoring of cells for apoptosis, cells can then be fixed and labeled with additional biomarkers of interest and imaged. These features render pSIVA-IANBD a particularly useful tool for toxicological studies of novel drugs in cell-based assays. pSIVA-IANBD is also suitable for flow cytometry and may supercede the widely used Annexin V-FITC PS probe which has has limited applications outside of flow cytometry.

Citation Format: Lisa S. Heiden, Stan Krajewski, Payton D. Quintel, Sujay K. Singh. Novel techniques for identifying apoptosis with pSIVA-IANBD in live and fixed 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 609. doi:10.1158/1538-7445.AM2013-609