bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract C122: The knockdown of SET protein modulates miRNAs and proteins levels involved in maintenance and progression of oral cancer.
Authors: Sousa Lucas O. , Sobral Lays Martin , Garcia Cristiana Bernadelli , Thomé Carolina Hassibe , Greene Lewis Joel , Leopoldino Andréia Machado
Published: August 2014

SET (TAF-1/I2PP2A) is a multifunctional protein that participates in cell cycle control, apoptosis, migration and histone acetylation. It has been reported to be increased in head and neck squamous cell carcinoma (HNSCC) and oral squamous cell carcinoma (OSCC). The participation of SET in the inhibitory complex of histone acetylation contributes to the maintenance of histone hypoacetylation, which presumably prevents transcription. The objective of the present study was to identify changes in the levels of miRNAs after the knockdown of the protein SET mediated by short hairpin RNA (shRNA) against mRNA coding for SET in the HNSCC cell line HN12 (HN12shSET) compared to its negative control (HN12shCTRL). After confirming the reduction of the amount of SET protein and the increase of histone acetylation, 88 miRNAs known to be related to several types of cancer were analyzed by quantitative real time PCR (PCR array - SYBR-Green®). The levels of miR-21, miR-133b, miR-137, miR-193a-3p, miR-15a, miR-29b, miR-100 and miR-125b, and others, were increased in HN12shSET cells. This increase was confirmed in HN12shSET cells and in the OSCC cell line Cal 27 with knockdown of SET using small interfering RNA against SET (siRNA; Cal 27siSET) after reverse transcription by quantitative real time PCR using TaqMan® assays. The knockdown of SET resulted in increased levels of Snail and vimentin proteins, characteristic of mesenchymal cells, and in reduced amounts of the epithelial cell marker protein, E-cadherin. These changes are characteristic of cells in epithelial-mesenchymal transition. Functional assays in transwell® chambers revealed that the HN12shSET cells migrate and invade more than HN12shCTRL cells. In contrast, transfection of pre-miR with a consequent increase of miR-29b (one miRNA up-regulated in HN12shSET) reduced cell migration and invasion in HN12shCTRL and HN12shSET lines, reversing the effect of the knockdown of SET on migration and invasion. The data indicate that the protein SET, which is accumulated in malignant cells, is involved in the regulation of miRNA levels and the modulation of the ability of migration, invasion and differentiation of HNSCC, in part by its action on the levels of proteins related to disease progression.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C122.

Citation Format: Lucas O. Sousa, Lays Martin Sobral, Cristiana Bernadelli Garcia, Carolina Hassibe Thomé, Lewis Joel Greene, Andréia Machado Leopoldino. The knockdown of SET protein modulates miRNAs and proteins levels involved in maintenance and progression of oral cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C122.
Abstract C139: Oncogenic ARAF mutation in a metastatic lung adenocarcinoma from a patient exhibiting sustained sorafenib response.
Authors: Greulich Heidi E. , Imielinski Marcin , Kaplan Bethany , Araujo Luiz , Amann Joseph , Horn Leora , Villalona Miguel , Meyerson Matthew , Carbone David
Published: August 2014

Targeted cancer therapies often induce "outlier" responses in molecularly defined patient subsets. One patient with advanced-stage lung adenocarcinoma who was treated with sorafenib demonstrated a near-complete clinical and radiographic remission for five years. Whole-genome sequencing and RNA sequencing on primary tumor and normal samples from this patient identified a somatic mutation, encoding ARAF S214C, present in the cancer genome and expressed at high levels. ARAF S214 binds 14-3-3 proteins when phosphorylated, and, by analogy to the paralogous residue of RAF1, presumably interferes with binding of ARAF to activated Ras. Mutation of S214 impaired the ability of ARAF to bind 14-3-3ξ, likely contributing to the oncogenic activity of ARAF S214C. Additional mutations affecting this residue of ARAF and a nearby residue in the related kinase RAF1 were demonstrated across 1% of an independent cohort of lung adenocarcinoma cases. The ARAF and RAF1 mutants transformed immortalized human airway epithelial cells and were associated with in vitro sorafenib sensitivity. These results suggest that mutant ARAF may be a novel oncogenic driver in lung adenocarcinoma and an indicator of sorafenib response.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C139.

Citation Format: Heidi E. Greulich, Marcin Imielinski, Bethany Kaplan, Luiz Araujo, Joseph Amann, Leora Horn, Miguel Villalona, Matthew Meyerson, David Carbone. Oncogenic ARAF mutation in a metastatic lung adenocarcinoma from a patient exhibiting sustained sorafenib response. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C139.
Abstract C142: Mortalin/HSPA9 regulates p21CIP1 expression in Raf/MEK/ERK-activated cancer cells.
Authors: Wu Pui Kei , Hong Seung-Keun , Veeranki Sudhakar , Karkhanis Mansi , Starenki Dmytro , Plaza Jose A. , Park Jong-In
Published: August 2014

Dysregulated Raf/MEK/ERK signaling is a common hallmark of tumorigenesis. However intriguingly, aberrant Raf/MEK/ERK activation triggers senescence-like growth arrest as a primary response in many cell types. This response is recognized as an innate tumor suppressive mechanism, which should be bypassed for Raf/MEK/ERK to drive carcinogenesis. Therefore, understanding of the bypass mechanisms is likely to provide an opportunity to design a novel therapeutic strategy. In this study, we report that mortalin, a member of the heat shock protein 70 (HSP70) family, has a role in bypassing the Raf/MEK/ERK-induced growth inhibition in cancer. Using tandem affinity purification and proteomic mass spectrometry, we found that mortalin is present in the MEK1/2 proteome. A specific physical interaction between mortalin and MEK was validated by co-immunoprecipitation analyses and in vitro binding assays of recombinant proteins. Immunohistochemical analysis revealed that mortalin is upregulated in human melanoma biopsies in correlation with tumor malignancy. Of note, mortalin levels were inversely correlated with p21CIP1 levels in a number of Raf/MEK/ERK-activated cancer cell lines, suggesting a role for mortalin in p21CIP1 regulation. Indeed, mortalin depletion elicited increased p21CIP1 transcription and MEK/ERK activation which were accompanied by G2/M phase cell cycle arrest and cell death in different B-Raf or K-Ras mutated cancer cell lines. Remarkably, blocking MEK/ERK activation, using the MEK1/2 inhibitor, AZD6244, dominant negative mutants of MEK or ERK, or RNA interference, abrogated p21CIP1 induction by mortalin depletion in cancer cells harboring B-RafV600E regardless of their p53 status. In contrast, mortalin overexpression suppressed B-RafV600E or ΔRaf-1:ER-induced MEK/ERK activation, p21CIP1 expression, and cell cycle arrest in cell types exhibiting normal MEK/ERK status. Other HSP70 family chaperones could not effectively replace mortalin for the observed effects on p21CIP1 regulation, suggesting a unique role for mortalin. These findings suggest that mortalin upregulation is a mechanism that underlies p21CIP1 silencing in Raf/MEK/ERK-activated cancer, identifying mortalin as a novel negative regulator of the pathway signaling. Mortalin may provide a target to reactivate the tumor suppressive signaling of Raf/MEK/ERK in cancer.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C142.

Citation Format: Pui Kei Wu, Seung-Keun Hong, Sudhakar Veeranki, Mansi Karkhanis, Dmytro Starenki, Jose A. Plaza, Jong-In Park. Mortalin/HSPA9 regulates p21CIP1 expression in Raf/MEK/ERK-activated cancer cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C142.
Abstract C143: Overcoming melanoma dual resistance to Vemurafenib and adoptive immunotherapy by chromatin remodeling drug.
Authors: Jazirehi Ali R. , Nazarian Ramin , Toress-Collado Antonio Javier , Economou James S.
Published: August 2014

BRAFV600E-inhibitors (BRAFi) and modern immune-based therapies have significantly improved the care of melanoma patients. We developed an in vitro model to determine whether tumor resistance mechanisms to this small molecule inhibitor of a driver oncogene and to cytotoxic T lymphocyte (CTL)-delivered apoptotic death signals were exclusive or intersecting. We generated melanoma sublines resistant to BRAFi Vemurafenib and to CTL recognizing the MART-1 melanoma antigen. Vemurafenib-resistant (VemR) sublines were cross-resistant to MART CTL indicating a common apoptotic pathway governing response to both modalities was disrupted. Pretreatment of VemR melanomas with a histone deacetylase inhibitor (HDACi) restored sensitivity to MART CTL apoptosis. These findings suggest that aberrant apoptotic pathways, amenable by a chromatin remodeling drug, regulate tumor resistance mechanisms and identify potential obstacles and solutions for combining these therapies clinically. TModulation of apoptotic machinery by inclusion of HDACi in BRAFV600E targeted therapy protocols as adjuvant will sensitize VemR (also CTL cross-resistant) human melanomas to immunotherapy.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C143.

Citation Format: Ali R. Jazirehi, Ramin Nazarian, Antonio Javier Toress-Collado, James S. Economou. Overcoming melanoma dual resistance to Vemurafenib and adoptive immunotherapy by chromatin remodeling drug. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C143.
Abstract C144: MAPK1 mutation E322K modulates ERK pathway output and feedback.
Authors: Guan Nin , Delpuech Oona , Greenawalt Danielle , Jacques Kelly , Gharahdaghi Farzin , Zinda Michael , Smith Paul J. , Guichard Sylvie M.
Published: August 2014

Mutations of MAPK1 E322K are observed in ∼8% of cervical ∼1% and head and neck cancer but there is currently little evidence of their impact on the ERK pathway. This study evaluated the impact of the E322K mutation on the ERK pathway output and feedback using an inducible system pTRIPZ in MCF10A immortalised cells. Expression of E322K MAPK1 following doxycycline induction reduced RSK phosphorylation on T359/S363 but increased pELK1 S383. DUSP6 protein levels were also increased in MCF10A expressing E322K mutant compared to WT. Pathway output was determined using the mRNA MEK signature (Dry et al. 2010). Following Dox induction, MCF10A E322K showed a 4-fold increase in DUSP2 mRNA and a 2-fold increase in DUSP6 mRNA while levels were unchanged in MCF10A expressing WT MAPK1. Conversely, >2-fold decrease in ETV5 levels was observed in E322K MCF10A compared to WT cells. Exposure of WT and E322K MCF10A cells to an ATP competitive ERK inhibitor showed a greater reduction in pRSK T359/S363 levels in E322K cells compared to WT cells (likely due to lower baseline levels) but abrogation of pELK1 required higher concentrations of the inhibitor in mutant compared to WT cells. Consistent with its mechanism of action, the ERK inhibitor increased pERK levels. Interestingly, the combination of the MEK inhibitor selumetinib with the ERK inhibitor completely abrogated pERK in WT but not in E322K mutant MCF10 cells. Overall, the results suggest that the E322K mutation increases ERK-regulated genes such as DUSP2 which may only partially de-phosphorylate ERK due to the E322K mutation affecting substrates’ binding to the D site. The impact of E322K on the sensitivity to ERK and MEK inhibition is currently under evaluation.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C144.

Citation Format: Nin Guan, Oona Delpuech, Danielle Greenawalt, Kelly Jacques, Farzin Gharahdaghi, Michael Zinda, Paul J. Smith, Sylvie M. Guichard. MAPK1 mutation E322K modulates ERK pathway output and feedback. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C144.
Abstract C147: Mechanism of resistance to a semisynthetic anticancer andrographolide derivative: Possible involvement of Ras-MAPK signaling pathway.
Authors: Stanslas Johnson , Wong Charng Choon , Sagineedu Sreenivasa Rao , Sidik Shiran , Sumon Shariful Hasan , Phillips Roger , Lajis Nordin Haji
Published: August 2014

SRJ09 (3,19-(2-bromobenzylidene)andrographolide) has been identified as a lead anticancer agent on the basis of its selectivity towards breast and colon cancers in the USA NCI 60 cancer cell lines screen (Jada et al (2008) Br J Pharmacol, 155(5):641-54). In our recent seminal publication (Hocker et al (2013), PNAS, 110(25):10201-6), we revealed that the compound is a binder of oncogenic mutant K-Ras. In the present investigation, HCT116 colon cancer cells were made resistant to SRJ09 to elucidate the mechanism of resistance. MTT cell viability assay was utilized to screen for cross-resistance to other analogues of andrographolide (AGP) and standard anticancer drugs. Flow cytometry technique was utilized for cell cycle study. Western blot was performed to determine the expression of cell cycle, apoptosis regulatory and cell signaling proteins. Additionally, reverse transcriptase (RT)-PCR was carried out to determine the cell cycle regulatory genes. SRJ09-resistant HCT116 displayed approximately 40-fold resistance towards SRJ09 compared with the parental cells. However, the cells were not resistant towards AGP analogues and standard anticancer drugs. The cells failed to arrest in G1 phase upon treatment with SRJ09, unlike the parental cells. RT-PCR confirmed the findings that protein expression of p21 was not up-regulated and CDK4 was not down-regulated, contrary to the parental cells. The resistance of the cells to undergo apoptosis upon treatment with SRJ09 was mainly attributed to the unchanged levels of Bax, Bid, caspase 8 and 9, otherwise activated in the parental cells. The inability of the cells to experience cell cycle arrest and apoptosis might be attributed in part to the marginal changes in phosphorylated c-Raf and ERK1/2 protein levels, which are in complete contrast to the parental cells. In conclusion, the mechanism of resistance is very likely due to failure of c-Raf and ERK1/2 to undergo dephosphorylation, implicating Ras-MAPK pathway as a target of SRJ09. This study was funded by Universiti Putra Malaysia Research University Grant Scheme (04-01-09-0713RU; 04-02-12-2017RU).

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C147.

Citation Format: Johnson Stanslas, Charng Choon Wong, Sreenivasa Rao Sagineedu, Shiran Sidik, Shariful Hasan Sumon, Roger Phillips, Nordin Haji Lajis. Mechanism of resistance to a semisynthetic anticancer andrographolide derivative: Possible involvement of Ras-MAPK signaling pathway. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C147.
Abstract C154: Kinetic analysis of the glutaminase activity of L-asparaginases derived from Erwinia chrysanthemi (Erwinase®) and Eshericia coli (Kidrolase).
Authors: Phillips Roger M. , Saleem Mohammed U. , Williams Adrian , Morgan Jon
Published: August 2014

Glutamine is an essential nutrient for cancer cells and increased glutaminolysis is widely recognised as a key feature of the metabolic reprogramming that occurs during malignant transformation. As a consequence of the role glutamine plays in cancer biology, targeting glutamine metabolism is of considerable interest with inhibition of the mitochondrial enzyme glutaminase showing promise in experimental models. An alternative or adjunct to the strategy of inhibiting glutamine metabolism intracellularly is to deplete extracellular pools of glutamine. L-asparaginases are known to possess glutaminase activity and the purpose of this study is to characterise the kinetics of the glutaminase activity of two clinically approved L-asparaginases, Kidrolase (derived from E. coli) and Erwinase (derived from Erwinia chrysanthemi). Glutaminase activity was measured using a two step reaction: initial reduction of glutamine to glutamate by Kidrolase/Erwinase followed by the NAD dependent conversion of glutamate to ammonia by L-glutamate dehydrogenase (GDH). Each reaction consisted of NAD (2mM), K2PO4 (50mM), GDH (3U), Erwinase or Kidrolase (10U), L-glutamine (concentrations ranged from 5mM to 1.25 μM) in a final volume of 1 ml Tris Acetate buffer (50mM, pH8.6). The reaction was started by the addition of Erwinase/Kidrolase and the formation of NADH monitored for 30 seconds at 340nm using a Cary UV/Vis spectrophotometer and a NADH molar extinction coefficient of 6,220 M-1cm-1. The reaction was linear between glutamine concentrations 100 and 1.25 μM. Using 10U in each reaction, Hanes Woolf kinetic plots generated Vmax parameters of 46.29 and 7.57 μM/min for Erwinase and Kidrolase respectively. Km values were 96.05 and 33.06 μM for Erwinase and Kidrolase respectively. Preliminary studies demonstrate that glutamine depletion induced a dose dependent reduction in cell proliferation in HCT116 p53+/+, HCT116 p53-/-, MDA-MD-231, MDA-MB-453, MCF7, TK10, A549 and T47D cell lines in vitro. In conclusion, these studies demonstrate that Erwinase has significant glutaminase activity and is superior to Kidrolase in terms of glutaminase activity. Erwinase is a good candidate for exploring the efficacy of glutamine depletion strategies and further studies are required to determine whether Erwinase can potentiate the activity of other approaches aimed at restricting the glutamine dependence of tumors.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C154.

Citation Format: Roger M. Phillips, Mohammed U. Saleem, Adrian Williams, Jon Morgan. Kinetic analysis of the glutaminase activity of L-asparaginases derived from Erwinia chrysanthemi (Erwinase®) and Eshericia coli (Kidrolase). [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C154.
Abstract C155: Targeting glucose metabolism to suppress Ph+B-ALL progression.
Authors: Liu Tingyu , Xiang Handan , Nichols Amanda , Gerriets Valerie , Kishton Rigel , Rizzieri David , Rathmell Jeffrey C.
Published: August 2014

Philadelphia chromosome positive B cell derived acute lymphoblastic leukemia (Ph+B-ALL) is a type of aggressive leukemia that lacks effective treatment. The high glucose metabolism observed in many other cancers suggests that targeting glucose metabolism may provide a novel therapeutic approach for Ph+B-ALL. However, metabolic features of Ph+B-ALL and metabolic stress responses have not been described. In this study, we examined human primary B-ALL samples, B-ALL cell lines and primary murine Ph+B-ALL to identify metabolic program and metabolic stress responses of Ph+B-ALL cells. Metabolic measurements of extracellular flux and glucose consumption showed that B-ALL cell lines are highly glycolytic and preferentially utilize glucose. Consistent with a dependence on elevated glycolysis, primary human B-ALL cells were more sensitive than normal B cells to inhibition of glycolysis with 2-deoxyglucose (2-DG). This metabolic stress induced cell death was likely to be mediated through p53 and Bcl-2 family proteins. 2-DG treatment induced expression of pro-apoptotic Bcl-2 family protein Bim and primary murine Ph+B-ALL cells generated from p53-/- or Bim-/- background remained viable even when treated with 2-DG. To specifically investigate how inhibition of glucose metabolism impacts B-ALL survival and disease progression in vivo with genetic tools, we generated murine Ph+ B-ALL cells on a Glut1fl/fl Ubi-CreER background that allow specific deletion of Glut1 in cancer cells upon treatment of tamoxifen. In vitro deletion of Glut1 reduced glucose uptake and glucose metabolism, although glycolysis was not wholly suppressed. This partial reduction of glucose metabolism led to greatly reduced cell proliferation and some cell death in vitro. Importantly, in vivo deletion of Glut1 after transfer of B-ALL cells into congenic recipients suppressed B-ALL progression and prolonged animal survival. These data show that B-ALL cells exhibit high glucose metabolism similar to other types of cancer and are sensitive to glucose metabolism inhibition. Reduction of glucose metabolism can impede B-ALL proliferation or cause cell death through p53 and Bim and thus suppress B-ALL progression in vivo.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C155.

Citation Format: Tingyu Liu, Handan Xiang, Amanda Nichols, Valerie Gerriets, Rigel Kishton, David Rizzieri, Jeffrey C. Rathmell. Targeting glucose metabolism to suppress Ph+B-ALL progression. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C155.
Abstract C165: Discovery of a lectin-like receptor family protein as a novel target for monoclonal antibody therapy.
Authors: Campagnoli Susanna , Parri Matteo , Grandi Alberto , De Camilli Elisa , Terracciano Luigi , Jin Boquan , Viale Giuseppe , Sarmientos Paolo , Grandi Guido , Pileri Piero , Grifantini Renata Maria
Published: August 2014

In our recent research activities, we identified a panel of novel candidate markers for prevalent cancers by a systematic tissue microarray analysis (TMA) of a large collection of polyclonal antibodies (approximately 1600) raised against membrane-associated and secreted human proteins currently marginally characterized. Of major interest were those proteins specifically detected in one or more of the five tumors with significant frequencies (data from 50 patients per each of the five cancers) and showing surface exposure on cancer cells. Among them, here we describe the molecular characterization of novel surface-associated protein belonging to the lectin-binding family proteins (involved in the innate immune response) for which very little knowledge exists. The protein was mainly over-expressed in breast, lung and ovary cancers (approximately 30-40% of tested cases) and it is involved in cell proliferation, migration and and invasiveness, as demonstrated by gene silencing using specific siRNA technology. Highly specific murine monoclonal antibodies (mAbs) were generated toward it are able to recognize the protein on the surface of breast and ovary cell lines. Of particular interest are two mAbs, which, upon binding, are efficiently internalized by breast and ovary cancer cells, indicating that they are amenable for the generation of antibody-drug conjugates. These two mAbs influence viability of breast and ovary cells in vitro. One of them shows antibody-dependent cell-mediated cytotoxicity in vitro on breast cancer cells. In vivo efficacy studies are ongoing to evaluate their therapeutic activity. Overall, data indicate that the lectin-binding protein and the specific mAbs here described could be developed as a novel tool for a targeted therapy alone or in combination with other treatments.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C165.

Citation Format: Susanna Campagnoli, Matteo Parri, Alberto Grandi, Elisa De Camilli, Luigi Terracciano, Boquan Jin, Giuseppe Viale, Paolo Sarmientos, Guido Grandi, Piero Pileri, Renata Maria Grifantini. Discovery of a lectin-like receptor family protein as a novel target for monoclonal antibody therapy. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C165.
Abstract C17: Genome-wide siRNA screen identifies diverse signaling pathways converging at mitochondria to regulate cell death.
Authors: Deng Jing , Letai Anthony
Published: August 2014

Programmed cell death (apoptosis) is regulated by the interplay of BCL2 family proteins, and by signals transduced to/from mitochondria. Recent studies in our group suggest that priming, a mitochondrial state involving interaction between anti- and pro-apoptotic proteins, can determine the sensitivity of chemotherapy. To understand how such priming status is regulated, we performed genome-wide siRNA screen using potent BCL2 inhibitor ABT737. The lead targets generated by the screen were validated and studied for potential mechanisms, revealing multiple signaling pathways that can regulate mitochondrial priming thus affect cell death. Knockdown of lead targets not only protects cells from ABT737 treatments, but also from other chemotherapeutics, such as paclitaxel and etoposide. The protection correlates with the protection of mitochondrial potential, suggesting that they may function upstream of mitochondrial outer membrane permeablization (MOMP). Targets initially identified in MDAMB231 breast cancer cell line were further validated in other cancer types, and demonstrated cell protection from chemo-treatments as well when down-regulated. Analysis of patients bearing deletions in genes or down-regulated RNA expression in some of these targets showed a trend to a poorer survival, supporting our finding that these targets may play important roles in regulating cell death and response to chemotherapy.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C17.

Citation Format: Jing Deng, Anthony Letai. Genome-wide siRNA screen identifies diverse signaling pathways converging at mitochondria to regulate cell death. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C17.
Abstract C186: Tyro3 is a potential target for the treatment of breast cancer.
Authors: Ekyalongo Roudy Chiminch , Mukohara Toru , Funakoshi Yohei , Tomioka Hideo , Kataoka Yu , Shimono Yohei , Toyoda Masanori , Kiyota Naomi , Fujiwara Yutaka , Minami Hironobu
Published: August 2014

Background: We previously identified the involvement of receptor tyrosine kinase Tyro3 in the cell growth mechanism of MCF-7 breast cancer cells. In the present study, we evaluated the potential of Tyro3 as a therapeutic target in various types of breast cancer cell lines.

Material and Methods: We compared outcomes following Tyro3 knockdown by siRNA in four estrogen receptor (ER)-positive/HER2-nonamplified (luminal-type; CAMA-1, T47D, MCF-7, and HCC1428), two ER-negative/HER2-amplified (HER2-type; HCC1419 and HCC1954), and two ER-negative/HER2-nonamplified (triple negative [TN]-type; MDA-MB-231 and HCC1143) breast cancer cell lines. The effect of Tyro3 knockdown on cell growth, cell cycle distribution, and cell signaling was evaluated by MTS assay, FACS, and Western blot, respectively.

Results: Tyro3 knockdown by siRNA induced the most prominent growth suppression in luminal-type cells, and to a lesser extent in HER2-type cells. In contrast, no growth inhibition following Tyro3 knockdown was observed in TN-type cells. The Tyro3 siRNA-induced growth inhibition in luminal-type cells was observed in both the estradiol (E2)-rich and -null conditions. Cellular sensitivity to Tyro3 siRNA was not correlated with baseline Tyro3 expression level or level of secretion of Tyro3 ligand Gas6. Phosphorylation of Tyro3 was detectable in five of six sensitive lines, but not in two resistant TN-type lines. The results of the growth assays significantly correlated with those of FACS analysis, with G0-G1/S cell cycle arrest observed only in Tyro3 siRNA-sensitive cells. Western blot analysis showed a decrease in phosphorylation of downstream signaling molecules, including ERK1/2, S6K, and STAT3, and in cyclin D1 in cell lines sensitive to Tyro3 siRNA only, and not in resistant TN-type cell lines.

Conclusions: Tyro3 is a potential therapeutic target in breast cancer, particularly in luminal-type cells. Phosphorylation of Tyro3 might be a predictive marker of sensitivity to Tyro3-targeted therapy.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C186.

Citation Format: Roudy Chiminch Ekyalongo, Toru Mukohara, Yohei Funakoshi, Hideo Tomioka, Yu Kataoka, Yohei Shimono, Masanori Toyoda, Naomi Kiyota, Yutaka Fujiwara, Hironobu Minami. Tyro3 is a potential target for the treatment of breast cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C186.
Abstract C19: Nelfinavir-induced autophagy and cell death as potential anticancer therapy for KSHV-associated lymphomas.
Authors: Kalu Nene , Biswas Sunetra , Shirley Courtney , Shamay Meir , Ambinder Richard F.
Published: August 2014

Kaposi sarcoma associated herpes virus (KSHV) is implicated in primary effusion lymphoma (PEL) and several other malignancies. Following infection, the virus evades the immune system by maintaining its genome in a latent state and only expressing few viral genes. Activation of KSHV lytic replication in vitro leads to viral lytic gene expression, the production of infectious virus and cell death. We previously showed that activation of herpes virus lytic replication can be achieved by treating PEL tumor cells with the proteasome inhibitor, bortezomib which induces endoplasmic reticulum (ER) stress. Here, we investigate the effects of nelfinavir, a FDA approved human immunodeficiency virus (HIV) protease inhibitor with anti-cancer activity. Anti-cancer effects of nelfinavir range from the induction of ER stress and apoptosis to Akt inhibition. Treatment of PEL cell lines with nelfinavir resulted in increased expression of ER stress markers: ATF4, CHOP and spliced XBP-1. Additionally, we observed nelfinavir-induced autophagy and cell death as indicated by an increase in LC-3B processing and a decrease in cell viability. We also observed the formation of autophagosomes by electron microscopy. In KSHV-infected tumor cells treated with nelfinavir, ER stress and autophagy precedes viral lytic gene expression. However, we show that while nelfinavir activates lytic gene expression, it also inhibits the export and cell-to-cell spread of infectious herpes viruses. At clinically relevant concentrations, nelfinavir induces autophagy and cell death in PEL cells while inhibiting virus export making it an attractive candidate for virus-associated cancer therapies.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C19.

Citation Format: Nene Kalu, Sunetra Biswas, Courtney Shirley, Meir Shamay, Richard F. Ambinder. Nelfinavir-induced autophagy and cell death as potential anticancer therapy for KSHV-associated lymphomas. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C19.
Abstract C190: A novel monoclonal antibody for colon cancer therapy.
Authors: Grandi Alberto , Campagnoli Susanna , Parri Matteo , De Camilli Elisa , Naldi Ilaria , Gherardini Lisa , Cinti Caterina , Terracciano Luigi , Jin Boquan , Sarmientos Paolo , Viale Giuseppe , Grandi Guido , Pileri Piero , Grifantini Renata Maria
Published: August 2014

In our recent research activities, we identified 89 novel candidate markers for prevalent cancers by a systematic Tissue microarray analysis (TMA) of a large collection of polyclonal antibodies (approximately 1600) raised against membrane-associated and secreted human proteins currently marginally characterized. Monoclonal antibodies were generated towards 20 distinct antigens that are being characterized and validated for diagnostic and therapeutic applications.

Here we describe a monoclonal antibody (mAb) targeting a protein of the protocadherin- family in colo-rectal cancer (CRC). The antibody specifically recognizes its target protein in cancer tissues, with concomitant negligible or marginal reactivity in normal colon. The protein was not previously described as CRC target. IHC data from CRC samples of approximately 200 patients, (collected at the bio-bank of the institute for Pathology of Basel, Switzerland and at the European Institute of Pathology of Milan, Italy) showed that the antibody binds up to 80% CRC cases (stages from 1 to 4) with high or moderate intensity. In most cases, the antibody gives plasma membrane by Immune Histochemistry (IHC). An expanded analysis on approximately 1000 CRC cases is ongoing to assess whether the membranous IHC staining associates with specific clinic-pathological parameters.

IHC analysis of 35 normal human tissues revealed that it gives intracellular staining in skeletal muscle, pituitary glands and prostate, whereas it gives background irrelevant staining in the other 32 tissues. The antibody is able to bind the surface of different colon cancer cells in vitro and in vivo. Upon binding it is efficiently internalized by colon cancer cells, suggesting that it can be exploited for the development of Antibody-Drug-Conjugate (ADC). Moreover, it inhibits tumor growth in athymic nude mice bearing HCT15 and HT29 colon cancer xenografts. Overall, data indicate that this monoclonal antibody could be developed for a targeted therapy of colo-rectal cancers, alone or in combinatorial strategies.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C190.

Citation Format: Alberto Grandi, Susanna Campagnoli, Matteo Parri, Elisa De Camilli, Ilaria Naldi, Lisa Gherardini, Caterina Cinti, Luigi Terracciano, Boquan Jin, Paolo Sarmientos, Giuseppe Viale, Guido Grandi, Piero Pileri, Renata Maria Grifantini. A novel monoclonal antibody for colon cancer therapy. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C190.
Abstract C204: GTPase/GAP/GEF-Glo™: A bioluminescent system to measure GTPase, GAP, and GEF activities.
Authors: Mondal Subhanjan , Goueli Said , Hsiao Kevin
Published: August 2014

It is well known that GTPases play major roles in cell functions such as GPCR signaling, cell proliferation, cell differentiation, cytoskeleton modulation, and cell motility. Deregulation or mutation of these proteins has considerable consequences resulting in multiple pathological conditions. Targeting GTPases and its regulators have been challenging due to lack of convenient assays. GTPases act as molecular switches cycling between an activated GTP-bound state (“ON”-state) and an inactive GDP-bound state (“OFF”-state). They have very high affinity for guanine nucleotides GDP or GTP and are extremely slow acting GTP-hydrolases. To mediate the process of shuttling between the active and inactive forms, GTPases are modulated by two groups of enzymes, guanine nucleotide exchange factors (GEF) and guanine nucleotide activating proteins (GAPs). The GEFs proteins substitute GTP for the bound GDP and thus activating them while the GAP family activates the hydrolysis of GTP and terminating their effector function. We have developed a homogenous bioluminescent assay for monitoring the enzyme activity of GTPase, GAP, and GEF proteins. Mg2+ plays a critical role in influencing affinity of GTPases with GTP/GDP, and manipulating Mg2+ concentrations in the reaction buffer allows continuous progression of the GTPase cycle and hydrolysis of GTP. The assay relies on the conversion of remaining GTP to ATP and detection of the latter using luciferin/luciferase combination. The assay system for measuring GTPase/GAP/GEF-Glo™ is enabling to monitoring the intrinsic GTPase-, GAP-stimulated GTPase-, GAP-, and GEF- activities. The assay system has a high dynamic range and signal-to-background ratio; it is in a convenient “add-mix-read” format and applicable to high throughput screening.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C204.

Citation Format: Subhanjan Mondal, Said Goueli, Kevin Hsiao. GTPase/GAP/GEF-Glo™: A bioluminescent system to measure GTPase, GAP, and GEF activities. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C204.
Abstract C217: Prognostic significance and molecular mechanism of ATP-Binding Cassette subfamily C member 4 in resistance to neoadjuvant radiotherapy of locally advanced rectal carcinoma.
Authors: Yu Zhiqi , Zhang Chang , Chai Rui , Du Yan , Gao Xianhua , Xing Junjie , Cao Guangwen , Fu Chuangang
Published: August 2014

Mechanism of radioresistance in rectal carcinoma remains largely unknown. The aim of this study is to verify the predictive value of ATP-Binding Cassette subfamily C member 4 (ABCC4) in locally advanced rectal carcinoma and explore possible molecular mechanisms by which ABCC4 confers the resistance to neoadjuvant radiotherapy. Methods: Expression of ABCC4 protein in pretreated biopsy tissue specimens from locally advanced rectal carcinoma patients was examined using immunohistochemistry. The 3-year overall survival and disease-free survival were used to determine long-term prognosis, and significant clinicopathological factors were identified by univariate log-rank test and multivariate Cox regression analysis. Lentivirus-mediated small hairpin RNA inhibited ABCC4 expression in colorectal carcinoma cell lines, and altered radiosensitivity was confirmed by xenograft model in vivo. Intracellular cyclic adenosine monophosphate concentration and cell cycle distribution following irradiation were further detected. Results: High expression of ABCC4 in pretreated tumors, p53 mutant type, high serum CEA and positive lymph node predicted an unfavorable postoperative prognosis for locally advanced rectal carcinoma patients after neoadjuvant radiotherapy in univariate analysis. Multivariate Cox regression analysis further demonstrated that high expression of ABCC4 and p53 mutant type were significant factors independently predicted an unfavorable long-term prognosis. Downregulation of ABCC4 expression significantly enhanced irradiation-induced suppression of tumor growth in xenograft model in vivo. Furthermore, inhibition of ABCC4 expression induced intracellular cyclic adenosine monophosphate accumulation and a noticeable deficiency of G1-S phase checkpoint in cell cycle following irradiation. Conclusions: Our study suggests ABCC4 as a novel predictive biomarker in predicting radioresistance and prognosis for locally advanced rectal carcinoma.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C217.

Citation Format: Zhiqi Yu, Chang Zhang, Rui Chai, Yan Du, Xianhua Gao, Junjie Xing, Guangwen Cao, Chuangang Fu. Prognostic significance and molecular mechanism of ATP-Binding Cassette subfamily C member 4 in resistance to neoadjuvant radiotherapy of locally advanced rectal carcinoma. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C217.
Abstract C227: High-throughput anchorage independence assay for screening inhibitors of cancer metastasis.
Authors: de Pril Remko , Oerlemans-Bergs Carla , Elstak Edo , Maghrani Ilhem , Janssen Richard
Published: August 2014

Tumor cells are capable of expanding without the requirement for cell adherence which is an indicator for their ability to form metastatic tumors in vivo. To do so, cells need to overcome cell cycle inhibitory signaling and/or apoptosis. This so-called anchorage-independent growth is an indicator of oncogenic transformation and deregulated cell growth and can be mimicked in vitro by culturing cells in soft agar. Our aim was to set up a high-throughput assay to identify inhibitors of anchorage-independent growth. Cancer cell lines were mixed with agar and seeded into 96-well plates. After polymerization, medium was added to the agar and subsequently refreshed twice a week. Depending on the cell line used the colonies in the agar were fixed after 8-14 days of culture and stained with phalloidin. The BD Pathway 435 was used to generate two collapsed stacks of 20 images per well. The inhibition of colony formation was analyzed using the Attovision software with an in-house developed algorithm. This approach was optimized using adenoviral knock-down constructs targeting known targets involved in anchorage independence as well as tool compounds which inhibited colony formation. This assay will be used for screening an adenoviral knock-down library to identify novel targets involved in anchorage independence as well as testing of compounds directed against the identified targets. We successfully developed an automated high-throughput anchorage independence assay in multiple cancer cell lines. This assay can be used for discovery and validation of novel targets and compounds for their potential to inhibit uncontrolled growth of cancer cells. Discovery of novel targets in metastasis may lead to the development of small molecule compounds or antibody therapeutics in cancer therapy.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C227.

Citation Format: Remko de Pril, Carla Oerlemans-Bergs, Edo Elstak, Ilhem Maghrani, Richard Janssen. High-throughput anchorage independence assay for screening inhibitors of cancer metastasis. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C227.
Abstract C229: Targeting STAT3 in prostate cancer: Identification of STAT3 as a direct target of the fungal metabolite galiellalactone.
Authors: Don-Doncow Nicholas , Escobar Zilma , Johansson Martin , Muñoz Eduardo , Sterner Olov , Bjartell Anders , Hellsten Rebecka
Published: August 2014

Introduction: The transcription factor Signal Transducer and Activator 3 (STAT3) is known to affect tumor growth in several malignancies. STAT3 is frequently found to be activated by phosphorylation (p-STAT3) in castration-resistant prostate cancer and constitutes a promising therapeutic target. The fungal metabolite galiellalactone (GL), a potent STAT3-signaling inhibitor, inhibits the growth, both in vitro and in vivo, of prostate cancer cells expressing activated STAT3 and inhibits growth and induces apoptosis of prostate cancer stem cell-like cells expressing p-STAT3. GL inhibits STAT3 signaling without blocking phosphorylation by a mechanism that we here aim to elucidate.

Materials and Methods: A biotinylated analogue of galiellalactone (GL-biot) was synthesized for identification of GL target proteins. The p-STAT3 expressing human prostate cancer cell line DU145 was incubated with GL-biot, followed by streptavidin beads to isolate proteins bound to GL-biot. Bound proteins were identified using Western blot analysis. Competition assays were performed by pre-incubating DU145 cell lysates with GL prior to GL-biot treatment. Localization of GL-biot and co-localization of GL-biot with STAT3 was assessed using confocal microscopy. Electrophoretic mobility shift assay (EMSA) was performed on GL treated DU145 cell lysates for analysis of STAT3 binding to DNA.

Results: The inhibitory activity of GL-biot on proliferation and inhibition of STAT3-signaling was similar to that of GL. By adding streptavidin beads to GL-biot-treated DU145 cell lysates, STAT3 was isolated and identified as a target protein. Pretreatment with GL prior to addition of GL-biot prevented the binding of GL-biot to STAT3 in a dose dependent manner. GL-biot was observed to interact also with STAT5 but not STAT1. Confocal microscopy revealed GL-biot in both the cytoplasm and nucleus of DU145 cells treated with GL-biot, appearing to co-localize with STAT3 in the nucleus. DU145 cell lysates subjected to EMSA with STAT3 probes showed a dose dependent decrease in STAT3-DNA binding when treated with GL.

Conclusions: Our results show that GL binds directly to STAT3 and inhibits DNA binding thus preventing the transcriptional activity of STAT3. This further validates GL as a promising STAT3-inhibitor for the treatment of castration-resistant prostate cancer.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C229.

Citation Format: Nicholas Don-Doncow, Zilma Escobar, Martin Johansson, Eduardo Muñoz, Olov Sterner, Anders Bjartell, Rebecka Hellsten. Targeting STAT3 in prostate cancer: Identification of STAT3 as a direct target of the fungal metabolite galiellalactone. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C229.
Abstract C237: Overcoming gemcitabine resistance with TRAIL plus enhancer of anti-survival potential in pancreatic cancer.
Authors: Lee Seungha , Kim So Y. , Kang Dongxu , Choi Hye J. , Kim Joo-Hang , Song Jae Jin
Published: August 2014

Although the combination of gemcitabine with a variety of cytotoxic and targeted agents has generally shown no significant survival advantage as compared with gemcitabine alone, still, many targeted agents against various pathways remain to be tested in pancreatic cancer. Previously, we have observed that the combined treatment with TRAIL plus shRNA of HSP27 induced complete cell death in MiaPaCa-2 pancreatic cell. Here three different targets were invited with TRAIL for chemotherapy combination with gemcitabine using three pancreatic cancer cell lines. We observed that TRAIL treatment to all three pancreatic cells have relieved gemcitabine-induced resistance. More importantly, three different pancreatic cancer cell lines showed their own unique differential sensitivities to targeted agents examined. For example, in case of MiaPaCa-2, gemcitabine/TRAIL/shRNA of survivin was more effective to cell death than HPAC, whereas TRAIL plus downregulation of survival-related molecules (survivin, XIAP, Bcl-xL) effectively enhanced cell death of Panc-1 even without gemcitabine. Then, we extended the survival-related molecules to upstreams such as Src, PI3K-Akt, or NF-κB and inhibited their downstream signals using PP2, wortmannin, SN50, respectively. From their results, TRAIL plus SN50 decreased NF-κB activation and concomitant significant cell death in MiaPaCa-2 which has an acquired resistance of NF-κB after gemcitabine. Based on this result, it was found that combination of gemcitabine/TRAIL/SN50 dramatically increased cell death of MiaPaCa-2, whereas in case of Panc-1 or HPAC, gemcitabine/TRAIL/PP2 dramatically increased cell death. Taken all together, a better knowledge of the multiple molecular pathways implicated in pancreatic carcinogenesis should be much more specifically considered to control the various gemcitabine-resistant pancreatic cancer cells.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C237.

Citation Format: Seungha Lee, So Y. Kim, Dongxu Kang, Hye J. Choi, Joo-Hang Kim, Jae Jin Song. Overcoming gemcitabine resistance with TRAIL plus enhancer of anti-survival potential in pancreatic cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C237.