bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract LB-025: Survivin is trafficked into cancer cell-derived exosomes via a microtubule-dependent mechanism
Authors: Kreger Bridget T. , Antonyak Marc , Cerione Richard
Published: August 2015

Cell-cell communication plays important roles in promoting tumor growth and metastasis, and these events are frequently targeted by therapies. The generation of extracellular vesicles (EVs), including exosomes and microvesicles (MVs), are beginning to be appreciated as one such form of communication that has important consequences in cancer. MVs are small vesicular structures (0.1-2 μm) that are derived from plasma membranes and are shed into the cell's surroundings; exosomes (30-100 nm) are rerouted endosomes that are released by the fusion of multi-vesicular bodies with the plasma membrane. Both types of EVs influence the behavior of recipient cells through the transfer of their cargo, which includes oncogenic factors, signaling proteins, RNA transcripts, and cytoskeletal components. However, the mechanism through which cargo is selectively trafficked to EVs has not yet been elucidated. We have discovered that disrupting microtubule dynamics in the MDAMB231 breast cancer cell line using the microtubule inhibitor nocodazole has the intriguing effect of significantly increasing the amount of the anti-apoptotic factor survivin that is contained within the EVs shed by these cells. This finding has been replicated in several additional human cancer cell lines and also when using the chemotherapy agent Paclitaxel (Taxol), which also inhibits microtubule dynamics. When MVs were separated from exosomes using procedures that were developed to resolve these two classes of EVs from MDAMB231 cell conditioned medium, survivin was found to be a specific cargo of exosomes and was absent from MVs. Moreover, the exosomes containing survivin enhanced the survival of recipient cells exposed to serum-starvation. Overall, these results show how a specific protein (e.g. survivin) can be selectively packaged into exosomes, as well as shed light on a novel mechanism that underlies Paclitaxel resistance.

Citation Format: Bridget T. Kreger, Marc Antonyak, Richard Cerione. Survivin is trafficked into cancer cell-derived exosomes via a microtubule-dependent mechanism. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-025. doi:10.1158/1538-7445.AM2015-LB-025
Abstract LB-026: Pin1 negatively regulated Grb7 protein stability via the ubiquitin-proteasome cascade requires the peptidyl-prolyl cis/trans isomerase activity of Pin1
Authors: Tai Yu-Ling , Shen Tang-Long
Published: August 2015

Growth factor receptor bound protein-7 (Grb7) is a multi-domain adaptor protein in cooperation with numerous tyrosine kinases to regulate various cellular signaling and functions. Although the regulatory mechanisms on the activation of Grb7 have been documented, the molecular mechanism governing Grb7 stability and its functional consequence is still not clearly understood. Here, we observed a novel negative regulatory mechanism of Grb7 by peptidyl-prolyl cis/trans isomerases Pin1 at the post-translational level. The Grb7 phosphorylation on the Ser194-Pro motif facilitated its binding to the WW domain of Pin1 and subsequently ubiquitination on Grb7, which, in turn, enhanced the process of proteosome-dependent proteolysis. Moreover, the interaction between Grb7 and Pin1 is dependent on the phosphorylation that is mediated by c-Jun N-terminal kinases MAPK. Both the phosphorylated Ser/Thr-Pro motif binding module and the peptidyl-prolyl cis/trans isomerase activity of Pin1 is essentail for Pin1-mediated Grb7 ubiquitin-proteasome proteolysis. By contrast, inhibition of Pin by lentiviral-mediated gene silencing resulted in accumulation of Grb7 protein as well as prolonged Grb7 protein stability. A stable Grb7S194A mutant that cannot be bound to and modulated by Pin1 utilize its potential to influence cell cycle progression. Our finding revealed that the Pin1/Grb7 complex formation enables negatively regulating Grb7-mediated cell proliferation due to the influence of Grb7 protein stability.

Note: This abstract was not presented at the meeting.

Citation Format: Yu-Ling Tai, Tang-Long Shen. Pin1 negatively regulated Grb7 protein stability via the ubiquitin-proteasome cascade requires the peptidyl-prolyl cis/trans isomerase activity of Pin1. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-026. doi:10.1158/1538-7445.AM2015-LB-026
Abstract LB-028: Cell density sensing alters TGF-beta signaling in a cell type-specific manner, independent from Hippo pathway activation
Authors: Nallet-Staub Flore , Yin Xueqian , Gilbert Cristèle , Marsaud Véronique , Ben Mimoun Saber , Javelaud Delphine , Leof Edward B. , Mauviel Alain
Published: August 2015

Cell-cell contacts inhibit cell growth and proliferation in part by activating the Hippo pathway that drives the phosphorylation and nuclear exclusion of the transcriptional coactivators YAP and TAZ. Cell density and Hippo signaling have also been reported to block TGF-β responses, based on the ability of phospho-YAP/TAZ to sequester TGF-β-activated SMAD complexes in the cytoplasm. Herein, we provide evidence that epithelial cell polarization interferes with TGF-β signaling well upstream and independent of cytoplasmic YAP/TAZ. Rather, polarized basolateral presentation of TGF-β receptors I and II deprives apically delivered TGF-β of access to its receptors. Basolateral ligand delivery nonetheless remains entirely effective to induce TGF-β responses. These data demonstrate that cell type-specific inhibition of TGF-β signaling by cell density is restricted to polarized epithelial cells and reflects the polarized distribution of TGF-β receptors, which thus impacts SMAD activation irrespective of Hippo pathway activation.

Citation Format: Flore Nallet-Staub, Xueqian Yin, Cristèle Gilbert, Véronique Marsaud, Saber Ben Mimoun, Delphine Javelaud, Edward B. Leof, Alain Mauviel. Cell density sensing alters TGF-beta signaling in a cell type-specific manner, independent from Hippo pathway activation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-028. doi:10.1158/1538-7445.AM2015-LB-028
Abstract LB-040: mTOR inhibitors induce apoptosis in colon cancer cells via CHOP-dependent DR5 induction upon 4E-BP1 dephosphorylation
Authors: He Kan , Zheng Xingnan , Li Mei , Zhang Lin , Yu Jian
Published: August 2015

The mammalian target of rapamycin (mTOR) is commonly activated in colon cancer. mTOR complex 1 (mTORC1) is a major downstream target of the PI3K/ATK pathway and activates protein synthesis by phosphorylating key regulators of mRNA translation and ribosome synthesis. Rapamycin analogs Everolimus and Temsirolimus are non-ATP-competitive mTORC1 inhibitors, and suppress proliferation and tumor angiogenesis and invasion. We now show that apoptosis plays a key role in their anti-tumor activities in colon cancer cells and xenografts through the DR5, FADD and caspase-8 axis, and is strongly enhanced by TRAIL and 5-fluorouracil. The induction of DR5 by rapalogs is mediated by the ER stress regulator and transcription factor CHOP, but not the tumor suppressor p53, upon rapid and sustained inhibition of 4E-BP1 phosphorylation, and attenuated by eIF4E expression. ATP-competitive mTOR/PI3K inhibitors also promote DR5 induction and FADD-dependent apoptosis in colon cancer cells. These results establish activation of ER stress and the death receptor pathway as a novel anticancer mechanism of mTOR inhibitors.

Citation Format: Kan He, Xingnan Zheng, Mei Li, Lin Zhang, Jian Yu. mTOR inhibitors induce apoptosis in colon cancer cells via CHOP-dependent DR5 induction upon 4E-BP1 dephosphorylation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-040. doi:10.1158/1538-7445.AM2015-LB-040
Abstract LB-043: Highly specific and sensitive nanoimmunoassay of PARP1 as cell death measurement toolkit
Authors: Pontikos Michael A. , Zimmermann Amos , Moncada Camilo , Abarca Heidemann Karin V. , Miller Jordan B. , Birabaharan Jonathan , Boyer Michael , Zuck Robin M. , Lorenzi Philip L. , Ascoli Carl
Published: August 2015

We developed and validated a highly specific toolkit to analyze PARP1 in a panel of control and siRNA knockdown cell lysates by multiple immunoassays. We can specifically and quantitatively distinguish PARP1 from 17 other PARP family members using highly specific antibodies and optimized methods.

Poly (ADP-ribose) polymerase-1 (PARP1) is a chromatin associated, ADP-ribosylating enzyme essential for multiple cellular functions, including

cardiac remodeling, vasoconstriction, regulation of astrocyte and microglial

function, long term memory, aging, transcription regulation, and DNA repair. More recently, it has been implicated in a new form of cell death termed parthanatos. PARP1 can also promote tissue survival by shifting the balance of cell death programs between autophagy and necrosis. Since PARP1 can promote tumorigenicity, it has gained traction as a therapeutic target in cancer. In that regard, clinical studies have shown vulnerability to PARP inhibitors in DNA repair defective cancers. One of the difficulties in analyzing PARP1 activity is the promiscuity of the reagents toward other PARP family members. Furthermore, the ability to examine

multiple tissue samples in parallel has been limited.

In this study we developed highly sensitive and specific antibodies against PARP1 fragments. Using siRNAs against 18 PARP family members, we validated antibody specificity by western blotting and nanoimmunoassay (NIA). NIA is a highly sensitive platform capable of screening up to 96 cancer samples at sub-microliter volume. Moreover, this platform permits quantitative analysis of non-modified PARP1 as well as post-translational modifications such as PARylation and phosphorylation using a single antibody for the measurement of all species. We anticipate this workflow will be amenable to a wide range of protein targets, ushering in a new frontier in diagnostic analysis.

Citation Format: Michael A. Pontikos, Amos Zimmermann, Camilo Moncada, Karin V. Abarca Heidemann, Jordan B. Miller, Jonathan Birabaharan, Michael Boyer, Robin M. Zuck, Philip L. Lorenzi, Carl Ascoli. Highly specific and sensitive nanoimmunoassay of PARP1 as cell death measurement toolkit. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-043. doi:10.1158/1538-7445.AM2015-LB-043
Abstract LB-047: Single-cell genomics reveals the genesis of cancer: copy number variation precedes single nucleotide variation
Authors: Xie Sunney , Huang Lei , Ma Fei , Wang Jingran , Ding Shigang , Gu Fang , Wang Wenjing , Zhang Jing
Published: August 2015

Recent advances in single cell whole genome amplification methods, such as MALBAC [1], have allowed accurate determination of copy number variation (CNV) as well as single nucleotide variation (SNV) of a single cancer cell [2]. In a bulk tissue sample at an early stage of cancer development, detection of abnormal CNV is often difficult, especially when the number of cells with abnormal CNV is small. Single cell genomic analysis is essential in evaluating the relation, if any, between CNV and SNV.

We have carried out for the first time single cell genomic analyses of colonoscopy biopsy at different adenoma stages. Some single cells in the Stage II adenoma have CNVs in the tumor suppresser gene APC (the copy number loss reduced from two to one) as well as SNVs in numerous cancer related genes. Interestingly, we found that while some single cells exhibited CNV reduction in APC without SNVs, all single cells with SNVs in COSMIC genes showed the CNV reduction in APC. Moreover we did not see a single cell that has SNVs but not CNVs in an adenoma. These data indicates that the CNV of APC precedes the SNVs in colon cancer development.

Furthermore, we found that the single cells from the same adenoma exhibited CNV patterns reproducible among all the cells, indicating that these cells were derived from the same stem cell, in which CNVs resulted from double strand breaks that could not be repaired perfectly.

Thus, we have established for the first time a correlation between SNVs and CNVs and propose that SNVs are generated as a consequence of abnormal CNVs in the genome. Therefore, our results could have a significant implication on the genesis of cancer.

References

(1) Zong, et al. Science 338: 1622-1626 (2012).

(2) Ni et al. PNAS 110, 21083 (2013).

Note: This abstract was not presented at the meeting.

Citation Format: Sunney Xie, Lei Huang, Fei Ma, Jingran Wang, Shigang Ding, Fang Gu, Wenjing Wang, Jing Zhang. Single-cell genomics reveals the genesis of cancer: copy number variation precedes single nucleotide variation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-047. doi:10.1158/1538-7445.AM2015-LB-047
Abstract LB-050: Patient-derived tumor xenografts in humanized NSG mice: a model to study immune responses in cancer therapy
Authors: Wang Minan , Keck James G. , Cheng Mingshan , Cai Danying , Shultz Leonard , Palucka Karolina , Banchereau Jacques , Bult Carol , Huntress Rick
Published: August 2015

Mouse models are frequently used to test the therapeutic efficacy of anti-cancer drugs. However, the translation of murine experimental data to treatments for patients with cancer often fails due to significant differences between the species, including the differences in the immune system. Our goal is to bridge this gap and to establish an in vivo preclinical model of human tumor immunotherapy by engrafting immunodeficient mice expressing a partial human immune system with human tumor implants. Humanized NOD-scid IL2Rγ (null) (hu-NSG) mice were initially generated by transplanting NSG mice with human CD34+ hematopoietic stem and progenitor cells (HSPCs) which support human hematopoietic and immune system development. Hu-NSG mice develop functional human T cells and B cells with high levels of TCR excision circles, complex TCR repertoire diversity and antigen-specific T cell proliferative responses. Several types of patient-derived tumors (non small cell lung cancer, sarcoma, triple negative breast cancer and invasive bladder cancer) were successfully implanted into HLA mismatched hu-NSG mice. Tumor growth curves show a delay in tumor growth in hu-NSG compared to non-humanized NSG mice. In a colon cancer xenograft model, treatment with chemotherapy agent (5-FU) or with a therapeutic antibody directed against VEGF (Avastin) resulted in decreased tumor growth. In addition to PDX tumors we have also tested human cancer cell lines. Tumor growth was observed in all hu-NSG mice implanted with human ovarian tumor cell line SKOV3-Luc-D3 cells at different time points post HSPC engraftment, showing no evidence of tumor rejection. Thus, our model of humanized mice bearing tumor-derived xenografts provides opportunities to study both the safety and efficacy of current cancer therapies.

Citation Format: Minan Wang, James G. Keck, Mingshan Cheng, Danying Cai, Leonard Shultz, Karolina Palucka, Jacques Banchereau, Carol Bult, Rick Huntress. Patient-derived tumor xenografts in humanized NSG mice: a model to study immune responses in cancer therapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-050. doi:10.1158/1538-7445.AM2015-LB-050
Abstract LB-054: Normal stem cell divisions, cancer incidence, and driver gene mutations
Authors: Tomasetti Cristian , Vogelstein Bert
Published: August 2015

Cancer arises through the sequential accumulation of mutations in oncogenes and tumor suppressor genes. We show that the incidence of many cancers is strongly correlated (0.80; P < 1.8 × 10-6) with the lifetime number of divisions of the normal stem cells maintaining that tissue's homeostasis. These results suggest that only ∼35% of the variation in cancer incidence among tissues is attributable to environmental factors or inherited predispositions. Moreover, we derive an approach to assess the evidence for environmental and inherited factors that contribute to a given cancer type vs. other cancer types.

The slope of the correlation noted above suggests that the number of driver gene mutations required to develop cancer must be smaller than what was previously estimated. We describe a novel approach to derive this estimate that combines conventional epidemiologic studies with genome-wide sequencing data. In two well-documented cancer types (lung and colon adenocarcinomas), we find that no more than three sequential mutations are needed.

Finally, we explore the role of chance and aging in acquiring the driver gene mutations required to develop cancer. We provide novel experimental evidence, in both mice and humans, of an unexpected relationship between the dynamics of division rates in normal tissues and age-specific cancer incidence. These data and analyses provide a firm basis for understanding the role of random, replicative mutations during stem cell division in cancer incidence.

Citation Format: Cristian Tomasetti, Bert Vogelstein. Normal stem cell divisions, cancer incidence, and driver gene mutations. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-054. doi:10.1158/1538-7445.AM2015-LB-054
Abstract LB-059: Opposing roles for protein tyrosine kinase 6 (PTK6) in colon cancer
Authors: Mathur Priya S. , Gierut Jessica J. , Xicola Rosa M. , Llor Xavier , Tyner Angela L.
Published: August 2015

Background: Protein Tyrosine Kinase 6 (PTK6, also called BRK) is a non-receptor tyrosine kinase expressed in differentiated epithelial cells of the skin, gastrointestinal tract, and prostate. Disruption of the Ptk6 gene led to impaired intestinal differentiation and increased intestinal proliferation in mice. However, PTK6 also has a tumor-promoting role in the colon, as disruption of the Ptk6 gene impaired carcinogen-induced STAT3 activation and tumorigenesis in mice. PTK6 also promotes survival of human colon cancer cell lines following DNA damaging treatments including γ-irradiation and chemotherapeutic drugs via STAT3 activation.

Results: We examined and manipulated colon cancer cell lines to understand these two seemingly contradictory roles for PTK6 in the colon. The human colon adenocarcinoma cell lines SW480 and SW620 represent a primary colon tumor and a metastasis from the same patient, respectively. PTK6 is highly expressed in SW480 cells and significantly reduced in SW620 cells. Active PTK6, phosphorylated on tyrosine residue 342, is not detectable in either cell line. Stable knockdown of PTK6 in SW480 cells results in an epithelial-to-mesenchymal transition (EMT), evidenced by decreased E-cadherin expression and increased ZEB-1 and Vimentin; as well as increased migration, wound-healing, and anchorage-independent growth of cells in culture. Additionally, knockdown of PTK6 led to increased in vivo tumorigenesis of xenograft cells in nude mice. Ectopic expression of wild-type PTK6 in SW620 cells drives the reverse process (MET), demonstrating a rescue of the EMT phenotype. When a constitutively active PTK6 construct is expressed in SW620 cells, it promotes activation of cytoplasmic oncogenic targets and downstream effectors, STAT3, FAK, BCAR1 and ERK5. Analysis of patient tissues demonstrates a downregulation of mRNA and protein levels, as well as a change in protein localization in tumor versus normal tissues; while PTK6 is detectable in the nuclei of differentiating normal cells it is excluded from nuclei in colon tumors.

Conclusions: Our studies indicate that PTK6 promotes the epithelial phenotype to antagonize the EMT in a kinase-independent manner and a kinase switch activates PTK6 to promote oncogenic signaling. PTK6 protein localization may function to regulate its specific role. Further examination of the complex role of this kinase could allow for a more nuanced understanding of colon cancer initiation and progression for the development of novel treatments.

Citation Format: Priya S. Mathur, Jessica J. Gierut, Rosa M. Xicola, Xavier Llor, Angela L. Tyner. Opposing roles for protein tyrosine kinase 6 (PTK6) in colon cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-059. doi:10.1158/1538-7445.AM2015-LB-059
Abstract LB-061: Bmi1 is required for the initiation of pancreatic cancer through an Ink4a-independent mechanism
Authors: Schofield Heather , Bednar Filip , Collins Meredith , Yan Wei , Zhang Yaqing , Shyam Nikhil , Eberle Jaime , Olive Kenneth , Bardeesy Nabeel , Nakada Daisuke , Simeone Diane , Morrison Sean , Pasca di Magliano Marina
Published: August 2015

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer related death in the United States. Many characteristic mutations in PDAC are known, but this information has so far failed to produce the development of effective treatments, highlighting the need for deeper understanding of the processes that drive tumorigenesis.

B-cell specific Moloney murine leukemia virus insertion site 1 (Bmi1), a Polycomb repressive group protein, is upregulated in PDAC and associated with poor prognosis. Our lab has shown that despite an oncogenic K-ras mutation, mice with pancreas specific loss of Bmi1 do not develop precancerous lesions, termed PanINs (Pancreatic Intraepithelial Neoplasia). This lack of PanIN development was also seen in the presence of pancreatitis, which is usually known to synergize with oncogenic K-ras to speed PanIN development. In other cancer types, Bmi1's effect on tumorigenesis was mechanistically linked to its regulation of the Ink4a/ARF genetic locus. However, we found that in PDAC, PanIN initiation was independent of Bmi1 control this locus. Further, impairment in the regulation of ROS generation was seen in vitro in pancreatic cancer cell lines lacking Bmi1. Regulating ROS generation is a vital step in the neoplastic process and has been shown in other systems to be controlled by Bmi1.

Overall, in this work we have shown that expression of the Polycomb group protein Bmi1 is necessary for the initiation of pancreatic precancerous lesions, and that the mechanism of Bmi1 requirement is independent of its repression of the Ink4a/ARF genetic locus. Given the recent pre-clinical development of a Bmi1 inhibitor, this work could provide rationale for future treatment of pancreatic cancer, a truly devastating disease.

Citation Format: Heather Schofield, Filip Bednar, Meredith Collins, Wei Yan, Yaqing Zhang, Nikhil Shyam, Jaime Eberle, Kenneth Olive, Nabeel Bardeesy, Daisuke Nakada, Diane Simeone, Sean Morrison, Marina Pasca di Magliano. Bmi1 is required for the initiation of pancreatic cancer through an Ink4a-independent mechanism. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-061. doi:10.1158/1538-7445.AM2015-LB-061
Abstract LB-071: Kub5-Hera/RPRD1B controls CDK1 regulation and synthetic lethality to PARP1 inhibition
Authors: Motea Edward A. , Fattah Farjana , Xiao Ling , Girard Luc , Rommel Amy , Morales Julio , Patidar Praveen , Porter Andrew , Minna John , Boothman David
Published: August 2015

Aberrant CDK1 regulation induces impulsive proliferation as well as genomic and chromosomal instability in cancer cells. However, the mechanism of how CDK1 is regulated at the transcriptional level, especially under pathological conditions (e.g. cancer), remains elusive. Here, we show that Kub5-Hera (K-H), a known transcription termination factor, plays a novel role in promoting CDK1 transcription to mediate DNA repair and cellular response to PARP1 inhibition in various cancer cell lines. At the molecular level, we demonstrate via luciferase reporter assays and mutagenesis studies that K-H binds the phospho-Serine 2-containing C-terminal domain (CTD) of RNA Pol II, which is then recruited to the cell-cycle homology region (CHR) of the CDK1 promoter to efficiently stimulate CDK1 transcription. Mechanistically, the loss of K-H concomitantly reduces CDK1 mRNA/protein expression levels, which consequently compromises homologous recombination (HR)-mediated DNA repair due to the lack of BRCA1 phosphorylation for efficient function. Intriguingly, loss of K-H stimulates PARP1 activity needed for cancer cell survival. Genetic and pharmacological ablation of PARP1 activity in BRCA-proficient cells with aberrant K-H reduction leads to synthetic lethality, which is reversed by re-expression of wild-type K-H but not the mutant with attenuated RNAPII binding. The mechanistic insights gained from this study should lay foundation toward promising new tumor biology-based targets (i.e., K-H as a novel therapeutic target) and innovative strategies to reduce normal tissue toxicity, increase tumor control, and expand the use of PARP1 inhibitors (e.g., Rucaparib) against BRCA-proficient cancers with aberrant K-H loss.

Citation Format: Edward A. Motea, Farjana Fattah, Ling Xiao, Luc Girard, Amy Rommel, Julio Morales, Praveen Patidar, Andrew Porter, John Minna, David Boothman. Kub5-Hera/RPRD1B controls CDK1 regulation and synthetic lethality to PARP1 inhibition. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-071. doi:10.1158/1538-7445.AM2015-LB-071
Abstract LB-074: The nucleolar ubiquitin-specific protease USP36 deubiquitinates and stabilizes c-Myc
Authors: Sun Xiao-Xin , He Xia , Yin Li , Sears Rosalie , Dai Mushui
Published: August 2015

c-Myc protein stability and activity are tightly regulated by the ubiquitin-proteasome system. Aberrant stabilization of c-Myc contributes to many human cancers. c-Myc is ubiquitinated by SCFFbw7 and several other ubiquitin ligases, whereas it is deubiquitinated and stabilized by ubiquitin-specific protease (USP) USP28. The bulk of c-Myc degradation appears to occur in the nucleolus. However, whether c-Myc is regulated by deubiquitination in the nucleolus is not known. Here, we report that the nucleolar deubiquitinating enzyme USP36 is a novel c-Myc deubiquitinase. USP36 interacts with and deubiquitinates c-Myc in cells and in vitro, leading to the stabilization of c-Myc. This USP36 regulation of c-Myc occurs in the nucleolus. Interestingly, USP36 interacts with the nucleolar Fbw7γ, but not the nucleoplasmic Fbw7α. Yet, it abolished c-Myc degradation mediated both by Fbw7γ and by Fbw7α. Consistently, knockdown of USP36 reduces the levels of c-Myc and suppresses cell proliferation. We further show that USP36 itself is a c-Myc target gene, suggesting that USP36 and c-Myc form a positive feedback regulatory loop. High expression levels of USP36 are found in a subset of human breast and lung cancers. Altogether, these results identified USP36 as a crucial and bono fide deubiquitinating enzyme controlling c-Myc's nucleolar degradation pathway.

Citation Format: Xiao-Xin Sun, Xia He, Li Yin, Rosalie Sears, Mushui Dai. The nucleolar ubiquitin-specific protease USP36 deubiquitinates and stabilizes c-Myc. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-074. doi:10.1158/1538-7445.AM2015-LB-074
Abstract LB-083: Regulation of the mitotic centrosome by heat shock protein 70
Authors: Fang Chieh-Ting , Kuo Hsiao-Hui , Yih Ling-Huei
Published: August 2015

A functional centrosome is essential for the assembly of a bipolar mitotic spindle and accurate chromosome segregation. Loss of centrosome integrity frequently occurs in transformed cells or in tumor tissues. Pathways maintaining the quality control of proteins are known to regulate the biogenesis and duplication of the centrosome. In this study, we explored the role of heat shock protein 70 (HSP70), an essential molecular chaperone for protein quality control, on maintaining the centrosome integrity during mitosis by treating cells with a HSP70 inhibitor or transducing cells with HSP70-specific shRNA. The effects of these treatments on mitosis progression, mitotic spindle assembly, and cell viability were investigated. Our results showed that inhibition or depletion of HSP70 disrupted microtubule nucleation and polymerization, induced abnormal mitotic spindles, and interfered with mitosis progression. In addition, HSP70 accumulated at the spindle pole and co-localized with γ-tubulin and pericentrin during mitosis. Loss of centrosomal HSP70 impeded the recruitment of pericentriolar components essential for centrosome maturation. These results indicate that HSP70 is required for the maintenance of a functional mitotic centrosome to support the assembly a bipolar mitotic spindle.

Citation Format: Chieh-Ting Fang, Hsiao-Hui Kuo, Ling-Huei Yih. Regulation of the mitotic centrosome by heat shock protein 70. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-083. doi:10.1158/1538-7445.AM2015-LB-083
Abstract LB-084: A role of long interspersed nuclear element-1 (LINE-1) for telomere maintenance in cells with alternative lengthening of telomeres
Authors: Aschacher Thomas , Wolf Brigitte , Kienzl Philip , Enzmann Florian , Messner Barbara , Holzmann Klaus , Bergmann Michael M.
Published: August 2015

LINE-1 elements (L1s, long interspersed nuclear elements-1) are abundant retrotransposons in the eukaryotic genome that are primarily known for facilitating aberrant recombination. Here we show the involvement of L1 in telomere maintenance in cells using the less characterized alternative lengthening of telomeres (ALT) mechanism. Interestingly, ALT cells show significantly higher expression levels of L1 when compared to TA cells. Knock-down (KD) of L1 in ALT cell lines was associated with reduced length of telomeres, an increase in telomere dysfunction foci, a reduced expression of ALT characteristics such as c-circles and ALT associated PML bodies, and a decreased growth. On the other hand, overexpression of L1 in ALT cell lines lead to a higher rate of c-circles and increased length of telomeres. LINE-1 not only bound to the C-strand of telomeric DNA but also to telomere-repeat-containing RNA (TERRA), a multifunctional component of human telomeres that is highly expressed in ALT cells and has been described to be involved in telomere stabilization. Whereas L1-KD decreased overall TERRA, L1 overexpression increased this RNA. Moreover, L1 KD abrogated the nuclear retention of TERRA. Moreover, the L1-ribonucleoprotein can use the polyadenylated form of TERRA as a template to generate telomere-specific DNA. Thus, L1 appears to contribute to telomere maintenance by a mechanism involving TERRA, either by ensuring its nuclear localization or by using this RNA as a template for the generation of telomere-specific DNA. Our findings now render L1 proteins a promising target in cancer therapy by interfering with telomere lengthening in ALT cells.

Citation Format: Thomas Aschacher, Brigitte Wolf, Philip Kienzl, Florian Enzmann, Barbara Messner, Klaus Holzmann, Michael M. Bergmann. A role of long interspersed nuclear element-1 (LINE-1) for telomere maintenance in cells with alternative lengthening of telomeres. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-084. doi:10.1158/1538-7445.AM2015-LB-084
Abstract LB-098: Antitumor studies: Design, synthesis, antitumor activity and molecular docking study of novel 2-deoxo-2-substituted-5-deazaalloxazines
Authors: Mahmoud Sawsan A. , Mohamed Mosaad S. , Abou Taleb Nageh A. , Nagamatsu Tomohisa , Ali Hamed I.
Published: August 2015

Cancer is the major threat to the public health worldwide, thereby we have a considerable interest to get potential antitumor agents via computational design, synthesis, functional elucidation, and biological evaluation of different deazaalloxazine analogs. Many of these compounds revealed higher selectivities against different tumor cell lines.

In the study the structure activity relationships (SAR) of the proposed derivatives was investigated, by applying structure based drug design (SBDD) using most advanced molecular modeling tool programs, namely: AutoDock 4.2 and Accelrys Discovery studio 2.0. These computational approaches aim to increase the speed and efficiency in the drug discovery process. The reasonable drug candidates were subjected to the chemical synthesis and biological in vitro test against different tumor cell lines. The docking study of the synthesized and the rationally designed derivatives was carried out using PTKs as target enzymes which was early reported as a proposed pathway for inhibition of cancer. The main outcome of this study is the synthesis of novel 2-methylthio, 2-(substituted alkyl amino), 2-(heterocyclic substituted), 2-amino, 2,4-dioxo and 2-deoxo-5-deazaalloxazine derivatives. Their antitumor activities against human T-cell acute lymphoblastoid leukemia cell line (CCRF-HSB-2), human oral epidermoid carcinoma cell line (KB), human breast cancer cell line (MCF-7) and human cervical cancer cell line (Hela) have been investigated in vitro. Many compounds showed promising antitumor activities. Furthermore, AutoDock study has been done by binding of the 5-deazaalloxazine analogs into c-kit PTK (PDB code: 1t46), where a good correlation between their IC50 and AutoDock binding free energy was exhibited.

Citation Format: Sawsan A. Mahmoud, Mosaad S. Mohamed, Nageh A. Abou Taleb, Tomohisa Nagamatsu, Hamed I. Ali. Antitumor studies: Design, synthesis, antitumor activity and molecular docking study of novel 2-deoxo-2-substituted-5-deazaalloxazines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-098. doi:10.1158/1538-7445.AM2015-LB-098
Abstract LB-099: A high-throughput screen of approved drugs uncovers a synergistic interaction targeting prostate cancer
Authors: Licciardello Marco P. , Markt Patrick , Klepsch Freya , Lardeau Charles-Hugues , Dürnberger Gerhard , Ivanov Vladimir , Colinge Jacques , Kubicek Stefan
Published: August 2015

Libraries of approved drugs contain some of the best studied small molecules and represent invaluable resources for drug repurposing and chemical biology studies. Access to comprehensive libraries of clinical compounds is limited and these collections are otherwise difficult to arrange. Using cheminformatics approaches we have assembled a non-redundant set of representative FDA-approved small molecules that we call the CeMM Library of Unique Drugs (CLOUD). The CLOUD covers the chemical and biological space of approved drugs, contains active forms of prodrugs, and can be screened at clinically relevant concentrations. In a combinatorial viability screen of the CLOUD we uncovered a synergistic interaction between two approved drugs. We show that the concomitant administration of these two small molecules induces apoptosis in the androgen receptor (AR)-dependent prostate cancer cell line LNCaP. Mechanistically, the combination affects AR signaling and the stability of the receptor itself. We are currently dissecting further aspects of the mechanism of action and performing in vivo experiments. Altogether, our data suggest that the combination of these two drugs can be repurposed for the treatment of prostate cancer.

Citation Format: Marco P. Licciardello, Patrick Markt, Freya Klepsch, Charles-Hugues Lardeau, Gerhard Dürnberger, Vladimir Ivanov, Jacques Colinge, Stefan Kubicek. A high-throughput screen of approved drugs uncovers a synergistic interaction targeting prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-099. doi:10.1158/1538-7445.AM2015-LB-099
Abstract LB-104: Excited electrons in melanin induce cyclobutane dimers in the dark
Authors: Premi Sanjay , Wallisch Silvia , Mano Camila , Weiner Adam , Bacchiocchi Antonella , Wakamatsu Kazumasa , Bechara Etelvino , Halaban Ruth , Douki Thierry , Brash Douglas E.
Published: August 2015

Sunlight-induced melanomas contain UV-signature mutations, which are caused by cyclobutane pyrimidine dimers (CPD). These photoproducts are typically created picoseconds after a UV photon is absorbed at adjacent thymines or cytosines. However, using immunohistochemistry, mass spectrometry, and RNAi, we find that melanocytes generate CPD for >3 hours after exposure to UVA or UVB, wavelengths found in sunlight and in tanning beds; these “dark CPD” constitute the majority of CPD induced. Using pharmacologic inhibitors, single-photon counting, and specific energy acceptors, we elucidated the mechanism. The process begins when UV-induced superoxide and nitric oxide combine to form peroxynitrite, one of the few biological molecules capable of exciting an electron. Excitation creates a quantum triplet state in the skin pigment melanin that has the energy of a UV photon but induces CPD by transferring its energy to DNA in a radiation-independent manner. Melanin is evidently carcinogenic as well as protective. These findings may underlie the dependence of UV-induced and spontaneous skin cancers on melanin type. The results also validate the long-standing suggestion that chemical generation of excited electronic states - the source of bioluminescence in lower organisms - is important in mammalian biology.

Citation Format: Sanjay Premi, Silvia Wallisch, Camila Mano, Adam Weiner, Antonella Bacchiocchi, Kazumasa Wakamatsu, Etelvino Bechara, Ruth Halaban, Thierry Douki, Douglas E. Brash. Excited electrons in melanin induce cyclobutane dimers in the dark. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-104. doi:10.1158/1538-7445.AM2015-LB-104
Abstract LB-105: Methanol extract of Cochrorus olitorus protects against potassium dichromate toxicity in albino rats
Authors: Osifeso Olabode O. , Akinwumi Kazeem A. , Adedoja Ayobami W.
Published: August 2015

Hexavalent chromate compounds are human carcinogens that pose great health hazard in several parts of the world especially in developing countries. In addition, an effective cure eludes the world. The study therefore evaluates the usefulness of the leafy vegetable and herb; Cochrorus olitorus (CO) against potassium chromate K2Cr2O7. Negative control animals were fed distilled water, while the positive control rats received K2Cr2O7 once per week. Test rats were exposed to 25, 50 and 100 mg/Kg body weight of CO alone for 42 days and / or 12 mg/kg body wt of K2Cr2O7 once per week before the animals were sacrificed. The frequency of micronucleated polychromatic erythrocytes (mPCEs) was monitored in bone marrow cells while aspartate aminotransferase (AST), alanine aminotransferase (ALT) and creatine levels were assessed in the serum. Haematological parameters were also monitored in test and control animals. The phytochemical analysis of CO was also carried out. K2Cr2O7 significantly (P<0.05) increased the levels of mPCE, AST, ALT, creatine, total white blood cells and lymphocytes as compared with the control. While percentage pack cell volume and neutrophils were reduced. In contrast, treatment with the different doses of CO restored the markers towards the levels of the negative control. Methanolic extract of CO is rich in flavonoids, saponins, anthraquinnones, terpernoid and phenols which may be responsible for the protection observed in this study. Our results suggest that methanol extract of CO has potentials in the treatment/management of chromate toxicity.

Note: This abstract was not presented at the meeting.

Citation Format: Olabode O. Osifeso, Kazeem A. Akinwumi, Ayobami W. Adedoja. Methanol extract of Cochrorus olitorus protects against potassium dichromate toxicity in albino rats. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-105. doi:10.1158/1538-7445.AM2015-LB-105
Abstract LB-106: Fusogenic-oligoarginine peptide-mediated silencing of the CIP2A oncogene suppresses oral cancer tumor growth in vivo
Authors: Alexander-Bryant Angela , Dumitriu Anca , Attaway Christopher , Yu Hong , Jakymiw Andrew
Published: August 2015

Intracellular delivery and endosomal escape of functional small interfering RNAs (siRNAs) remain major barriers limiting the clinical translation of RNA interference (RNAi)-based therapeutics. Recently, we demonstrated that a endosome-disruptive peptide we synthesized termed, 599, could enhance the intracellular delivery and bioavailability of siRNAs designed to target the CIP2A oncoprotein (siCIP2A) into oral cancer cells and consequently inhibit oral cancer cell invasiveness and anchorage-independent growth in vitro. Thus, to further assess the therapeutic potential of the 599 peptide in mediating RNAi-based therapeutics for oral cancer and its prospective applicability in clinical settings, the objective of the current study was to determine whether intratumoral dosing of the 599+siCIP2A complex could induce silencing of CIP2A and consequently impair tumor growth using a xenograft oral cancer mouse model. Our results demonstrated that the 599 peptide was able to protect siRNAs from degradation by serum and ribonucleases in vitro, confirming the stability of the 599+siRNA complex and its potential for in vivo utility. Moreover, 599 peptide-mediated delivery of siCIP2A to tumor tissue induced CIP2A silencing without any associated toxicity, consequently resulting in reduction of the mitotic index and significant inhibition of tumor growth. Together, these data suggest that the 599 peptide carrier could be a clinically effective mediator of RNAi-based cancer therapeutics.

Citation Format: Angela Alexander-Bryant, Anca Dumitriu, Christopher Attaway, Hong Yu, Andrew Jakymiw. Fusogenic-oligoarginine peptide-mediated silencing of the CIP2A oncogene suppresses oral cancer tumor growth in vivo. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-106. doi:10.1158/1538-7445.AM2015-LB-106
Abstract LB-124: Tumor-educated platelets allow for multiclass liquid biopsy-based diagnosis of cancer
Authors: Best Myron , Sol Nik , Kooi Irsan , Nilsson Jonas , Westerman Bart , Ylstra Bauke , Dorsman Josephine , Smit Egbert , Verheul Henk , Reijneveld Jaap , Tannous Bakhos , Wesseling Pieter , Würdinger Thomas
Published: August 2015

Background: Cancer diagnosis is frequently hampered by limited access to adequate tissue of the primary tumor or of metastatic lesions. To overcome such limitations, the use of blood-based liquid biopsies has been suggested. Blood represents a biosource of tumor-educated platelets (TEPs) that sequester biomolecules during tumor growth, thereby altering the platelet mRNA profile.

Methods: Blood platelet samples of 175 patients with cancer covering five tumor types (40 non-small cell lung cancer, 39 glioblastoma, 37 colorectal cancer, 35 pancreatic cancer, and 24 breast cancer) and of 52 healthy donors were isolated from whole blood by differential centrifugation. Total RNA was isolated, subjected to SMARTer mRNA amplification and submitted for whole transcriptome mRNA sequencing on the Illumina platform. Healthy donors, pan-cancer, and individual cancer classes were distinguished by a self-learning support vector machine (SVM) algorithm, using transcripts with moderate to high expression.

Results: The 227 blood platelet samples were successfully sequenced and demonstrated a good intersample correlation of the detected mRNAs. Based on mRNA profiles, all tumor samples were clearly distinguished from healthy individuals: the pan-cancer SVM-supported classification test reached a sensitivity of 97% and a specificity of 92% to distinguish cancer patients from healthy controls. Also, all patients without overt metastases were correctly predicted as cancer patients. Moreover, a multiclass cancer diagnostics TEP-test, to distinguish multiple tumor subclasses and healthy controls provided an overall accuracy of 70%, far exceeding random classification. In addition, we distinguished HER2-positive, and mutant KRAS and EGFR tumors from their wild-type counterparts. Also, patients with metastatic tumors in lung, brain, and liver were accurately diagnosed according to the tumor in the tissue of origin.

Conclusion: Molecular interrogation of TEP-based liquid biopsies may leverage cancer diagnostics. TEPs provide a circulating biosource for pan-cancer, multiclass, and molecular cancer classification. Of interest, this tool might also allow for blood-based highly sensitive early-stage cancer screening.

Citation Format: Myron Best, Nik Sol, Irsan Kooi, Jonas Nilsson, Bart Westerman, Bauke Ylstra, Josephine Dorsman, Egbert Smit, Henk Verheul, Jaap Reijneveld, Bakhos Tannous, Pieter Wesseling, Thomas Würdinger. Tumor-educated platelets allow for multiclass liquid biopsy-based diagnosis of cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-124. doi:10.1158/1538-7445.AM2015-LB-124