bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract B64: The role of JAK1/2-STAT3 as acute resistance mechanism to MEK inhibition in BRAF mutant colorectal cancer.
Authors: Carson Robbie , Celtikci Basak , Dunne Philip , Johnston Patrick , Van Schaeybroeck Sandra
Published: August 2014

Background: Oncogenic mutations in BRAF occur in 8% of patients with advanced colorectal cancer (CRC) and have been shown to correlate with poor prognosis. In contrast to BRAF mutant (MT) melanoma, where the BRAF inhibitor PLX4032 has shown significant increases in response rates and overall survival compared to standard Dacarbazine treatment, only minor responses to PLX4032 treatment have been reported in BRAFMT CRC. Clear understanding of the vulnerabilities of BRAFMT CRC is important, and identification of druggable targets uniquely required by BRAFMT CRC tumors has the potential to fill a gap in the therapeutic armamentarium of advanced CRC. The aim of this study was to identify novel resistance mechanisms to MAPK inhibition in BRAFMT CRC.

Methods: Paired BRAFMT/WT RKO and VACO432 CRC cell line models and non-isogenic BRAFMT LIM2405, WiDR and COLO205 CRC cells were used. Changes in protein expression/activity were assessed by Western Blotting. Interaction between MEK1/2 and JAK1/2 inhibition was assessed using the MTT cell viability assays and flow cytometry. Apoptosis was measured using Western blotting for PARP, cleaved caspase 3/8 and caspase 8, 3/7 activity assays.

Results: Treatment with MEK1/2 inhibitors AZD6244, GSK1120212, UO126 and PD98059 resulted in acute increases in STAT3 activity in the BRAFMT RKO and VACO432 cells but not in their BRAFWT clones and this was associated with increases in JAK2 activity. Inhibition of JAK/STAT3 activation using gene specific siRNA or small molecule inhibitors TG101348 or AZD1480, abrogated this survival response and resulted in significant increases in cell death when combined with MEK1/2 inhibitors AZD6244 or GSK1120212 in BRAFMT CRC cells. In addition, combination of MEK1/2 and JAK/STAT3 inhibition resulted in strong synergy with CI values between 0.3 and 0.7 in BRAFMT CRC cells.

Conclusions: We have identified JAK/STAT3 activation as an important escape mechanism for BRAFMT CRC following MEK1/2 inhibition. These data provide a strong rationale for further investigation of combination of MEK1/2 and JAK/STAT3 inhibition in BRAFMT in vivo models.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B64.

Citation Format: Robbie Carson, Basak Celtikci, Philip Dunne, Patrick Johnston, Sandra Van Schaeybroeck. The role of JAK1/2-STAT3 as acute resistance mechanism to MEK inhibition in BRAF mutant colorectal 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 B64.
Abstract B69: Dynamic reprogramming of signaling following Met inhibition reveals pathways for cell proliferation and negative feedback in gastric cancer.
Authors: Lai Andrea , Cory Sean , Zhao Hong , Sehat Bita , Tofigh Ali , Guiot Marie-Christine , Thompson Crista , Bell Emily S. , Bertos Nicholas , Ferri Lorenzo E. , Hallett Michael , Park Morag
Published: August 2014

The Met receptor tyrosine kinase activates multiple signalling pathways in response to stimulation by ligand. However, the signalling events that occur under chronic Met activation in cancer cells are poorly understood. Utilizing a pharmacological small-molecule inhibitor of Met, we demonstrate that the gastric cancer cell lines, Okajima, MKN45, Snu-5 and KATO II are dependent on Met signalling for proliferation and anchorage-independent growth. We show that short-term inhibition of Met leads to waves of changes in gene expression such as a rapid loss of immediate-early genes, negative regulators, and a more delayed decrease in genes involved in the cell-cycle and proliferation. At the post-translational level, the Ras-Erk, PI3K-Akt and STAT3 pathways were all found to be dependent on Met signalling in these cells, though STAT3 signalling was the key requirement for proliferation, downstream from Met. We also observed the loss of negative regulators of Erk signalling, such as DUSP4 and DUSP6, which allowed for Mek-dependent reactivation of Erk, in the presence of Met inhibitor. As Met inhibitors are currently in clinical trials, it is essential to understand the molecular events that occur upon treatment, such that we can more effectively target sensitive tumours and avoid resistance.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B69.

Citation Format: Andrea Lai, Sean Cory, Hong Zhao, Bita Sehat, Ali Tofigh, Marie-Christine Guiot, Crista Thompson, Emily S. Bell, Nicholas Bertos, Lorenzo E. Ferri, Michael Hallett, Morag Park. Dynamic reprogramming of signaling following Met inhibition reveals pathways for cell proliferation and negative feedback in gastric 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 B69.
Abstract C118: TERT promoter mutations in human gliomas.
Authors: Lima Jorge , Reis Rui M. , Catarino Telmo , Peixoto Joana , Vinagre Joao , Batista Rui , Castro Ligia , Pardal Fernando , Sobrinho-Simoes Manuel , Maximo Valdemar , Soares Paula
Published: August 2014

Reactivation of telomerase has been implicated in human tumorigenesis, but the underlying mechanisms remain poorly understood. Recently, two mutations (-124G>A and -146G>A) in the promoter region of the telomerase catalytic subunit (TERT) were found in a significant proportion of human tumors, including gliomas, thyroid carcinomas, bladder carcinomas and melanomas. These mutations resulted in increased expression of telomerase, both in vitro and in vivo. In order to further characterize the prevalence of TERT mutations in human tumors, we screened a cohort of 273 human gliomas and found an overall TERT mutation frequency of 50%. TERT mutations were most commonly found in high grade tumors such as glioblastoma multiforme (GBM) and gliosarcoma (65% and 89%, respectively), whereas low grade tumors such as pilocytic astrocytoma and diffuse astrocytoma showed a low frequency of TERT mutations (6% and 16%, respectively). Oligodendrogliomas (grade II) and anaplastic oligodendrogliomas (grade III) also harbored a high frequency of TERT mutations (44% and 52%, respectively). In GBM patients, the presence of TERT mutations was significantly associated with a higher mean age at diagnosis (p<0.0001) and worse survival (p=0.006). Our results show that TERT promoter mutations are frequent in human GBM and oligodendrogliomas and are associated with worse survival in GBM patients.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C118.

Citation Format: Jorge Lima, Rui M. Reis, Telmo Catarino, Joana Peixoto, Joao Vinagre, Rui Batista, Ligia Castro, Fernando Pardal, Manuel Sobrinho-Simoes, Valdemar Maximo, Paula Soares. TERT promoter mutations in human gliomas. [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 C118.
Abstract C135: AZ’4425 is a potent, selective, and orally bio-available progesterone receptor antagonist that has shown anti-tumor activity and inhibition of cancer stem cell proliferation.
Authors: Chresta Christine M. , Alferez Denis , Cerillo Georgia , Still Emma , Hughes Adina , Walker Graeme , Kendrew Jane , Sadler Claire , Harris Jayne , Simpson Iain , Thomas Andrew P. , Rabow Al , Clarke Robert , Howell Sacha , Richmond Graham
Published: August 2014

Progesterone is an important hormone in breast cancer; the paracrine mediator of the progesterone receptor, RANKL plays a critical role in cancer stem cells, metastasis and tumorigenesis. Steroidal inhibitors of PR have shown modest clinical activity but have been limited by toxicity. AZ’4425 is a novel, potent non-steroidal progesterone receptor (PR) antagonist which selectively inhibits PR compared to other steroid hormone receptors (∼ 1000-fold ER, MR, GR, AR). AZ’4425 competitively inhibits progesterone binding, thus preventing progesterone-induced phosphorylation of PR, nuclear translocation and PR-induced transcription with an IC50 of 26nM. Anchorage independent growth of T47D cells is similarly inhibited with an IC50 of 25nM. Using cancer stem cell (CSC) assays (mammosphere formation and ALDH positivity) we have shown broad cellular activity in both ER/PR positive tumor cell lines and early and late stage clinical samples. Furthermore, AZ’4425 prevents the increase in CSCs, and miRNA changes (miR221/2 and miR200c) induced by anti-estrogen therapies, suggesting a potential role in prevention of acquired resistance. In vivo, the PR antagonist inhibits transcription of progesterone-induced genes including RANKL and SGK1. Notably, AZ’4425 results in significant inhibition of new tumor formation and results in stasis and regression of DMBA-induced tumors, both as monotherapy and in combination with letrozole.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C135.

Citation Format: Christine M. Chresta, Denis Alferez, Georgia Cerillo, Emma Still, Adina Hughes, Graeme Walker, Jane Kendrew, Claire Sadler, Jayne Harris, Iain Simpson, Andrew P. Thomas, Al Rabow, Robert Clarke, Sacha Howell, Graham Richmond. AZ’4425 is a potent, selective, and orally bio-available progesterone receptor antagonist that has shown anti-tumor activity and inhibition of cancer stem cell proliferation. [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 C135.
Abstract C219: Novel MET/FGFR/AXL kinase inhibitor S49076 exerts radiosensitizing activity in vitro and in vivo.
Authors: Chargari Cyrus , Clémenson Céline , Mondini Michele , Ferte Charles , Jacquet-Bescond Anne , Cattan Valérie , Depil Stéphane , Deutsch Eric
Published: August 2014

Background: S49076 was developed as an oral ATP-competitive inhibitor of MET receptor and of other kinases involved in tumor development. It is being assessed as single agent in phase I study. We examined its potential as a radiosensitizer in non-MET dependent tumor models.

Experimental Procedures: Cell viability and clonogenic survival assays were performed in two non small-cell lung carcinoma (NSCLC) cells (H460 and A549) and in breast cancer cells MDA-MB-231. Then, the drug was tested in combination with ionizing irradiation (IR) in vitro. The combination was assessed in subcutaneous tumor xenografts and in NSCLC orthotopic models of luciferine-expressing H460luc and A549luc tumors. Immunohistochemical correlates were performed and the activity of S49076 was assessed in a lung metastases model of intravenous injections of A549 cells.

Results: The drug as single agent inhibited proliferation (IC50 ranging from 0.50 μM to 1.1 μM) and clonogenic survival (IC50 ranging from 0.34 μM to 0.86 μM). Transcriptomic analyses showed that antitumor effect was associated with modulation of molecular pathways involved in radiation response (p53 and ATM). S49076 and IR inhibited synergistically clonogenic survival in H460 cells: sensitivity enhancement ratio at 0.6 μM was 2 for a single radiation dose of 2 Gy. Combination was additive in A549 and MDA-MB-231 cells. S49076 at 50 mg/kg twice daily enhanced radiation-induced growth delay in subcutaneous tumor xenografts, accompanied with an increase in TUNEL staining. Dose enhancement factors were 3.2 and 1.6 in H460 and A549 tumors, respectively. In lung orthotopic models, S49076 generated significant delay in time course of bioluminescent signal and improved median estimated survival, with significant benefit of combination over either IR or S49076 alone. Finally, treatment with S49076 improved significantly median survival in the lung metastases model (14 versus 22 days, p < 0.001).

Conclusions: Altogether, these data suggest that S49076 has a potential as radiosensitizer in non MET-dependent cell lines, and that this effect is accompanied with a modulation of apoptosis.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C219.

Citation Format: Cyrus Chargari, Céline Clémenson, Michele Mondini, Charles Ferte, Anne Jacquet-Bescond, Valérie Cattan, Stéphane Depil, Eric Deutsch. Novel MET/FGFR/AXL kinase inhibitor S49076 exerts radiosensitizing activity in vitro and in vivo. [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 C219.
Abstract CN06-01: Cancer stem cells and the EMT.
Authors: Weinberg Robert A.
Published: August 2014

The epithelial-mesenchymal transition (EMT) is a cell-biological program that enables both normal and neoplastic epithelial cells to acquire mesenchymal characteristics. In the case of carcinoma cells, this enables them to invade and to disseminate. In addition, once disseminated, the EMT enables carcinoma cells to extravasate and to establish an initial foothold in the parenchyma of distant organs. Of note, the EMT may be triggered in carcinoma cells by signals that they receive from the adjacent activated stroma, and the proclivity of primary carcinoma cells to activate the EMT program in response to such signals may be an indication of their eventual tendency to invade and metastasize. In addition, activation of the EMT program empowers carcinoma cells to enter into the stem cell state, as defined by their tumor-initiating potential. Importantly, however, cells that have passed through an entire EMT program and become entirely mesenchymal are not qualified to enter into the CSC state and seed metastases. Hence, partial activation of this program would be appear to be optimal for entrance into this state and acquisition of tumor-initiating potential.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):CN06-01.

Citation Format: Robert A. Weinberg. Cancer stem cells and the EMT. [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 CN06-01.
Abstract PL02-01: The metabolic phase of the cell cycle.
Authors: Gottlieb Eyal
Published: August 2014

Due to the nature of tumorigenesis, cancer cells constantly encounter environments in which nutrient and oxygen availability is severely compromised. In order to survive these harsh conditions, cancer cell transformation is often coupled with large changes in metabolism necessary to satisfy the exigencies for energy and biomass imposed by continued cellular proliferation. These variations often involve increases in the consumption and metabolism of extracellular resources, such as glucose, amino acids, and lipids. We were particularly interested in investigating the role of lipid metabolism in cancer cell growth. A siRNA screen against a panel of genes involved in lipid metabolism, specifically identified acetyl-coA synthetase as a critical enzyme for the growth of breast and prostate cancer cells. Bioinformatics analyses revealed that acetyl-coA synthetase 2 (ACSS2) was indeed amplified in a subset of invasive breast carcinomas. Furthermore, we characterized the contribution of acetate to lipid metabolism through liquid chromatography/mass spectrometry-based metabolomics and lipidomic approaches. Induced silencing of ACSS2 reduced cell growth of both breast and prostate cancer cells and revealed a heretofore unappreciated role of acetate as an anabolic precursor for de novo lipid biogenesis and cell growth.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):PL02-01.

Citation Format: Eyal Gottlieb. The metabolic phase of the cell cycle. [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 PL02-01.
Abstract PL07-04: Engineered T cell transfer for cancer immunotherapy in the era of synthetic biology: Challenges and opportunities.
Authors: June Carl H.
Published: August 2014

Adoptive T cell transfer for cancer and chronic infection is an emerging field that shows promise in recent trials. Synthetic-biology-based engineering of T lymphocytes to express high-affinity antigen receptors can overcome immune tolerance, which has been a major limitation of previous immunotherapy-based strategies. Advances in cell engineering and culture approaches to enable efficient gene transfer and ex vivo cell expansion have facilitated broader evaluation of this technology, moving adoptive transfer from a "boutique"’ application to the cusp of a mainstream technology. Recent observations from ongoing clinical trials in patients with leukemia and carcinomas will be discussed. The major scientific challenge currently facing the field is identification of cancer specific targets to avoid on-target, off-tissue toxicity. As the field of adoptive transfer technology matures, the major engineering challenge is the development of automated cell culture systems, so that the approach can extend beyond specialized academic centers and become widely available.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):PL07-04.

Citation Format: Carl H. June. Engineered T cell transfer for cancer immunotherapy in the era of synthetic biology: Challenges and opportunities. [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 PL07-04.
Abstract PR08: Loss of oncogenic Notch1 signaling contributes to PI3 kinase inhibitor resistance in T lineage leukemia.
Authors: Dail Monique
Published: August 2014

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive cancer for which current therapies are toxic and frequently ineffective. Mutations that activate the Notch1 and Ras/PI3K/Akt signaling pathways are common in T-ALL and often coexist. Thus, inhibiting these pathways alone and in combination is a rational therapeutic strategy. Notch1 inhibition with a gamma-secretase inhibitor (GSI) was recently evaluated in patients with disappointing results. PI3K inhibitors are just entering Phase I clinical trials. GDC-0941 is a potent PI3K inhibitor that is currently advancing in clinical development that we evaluated for efficacy and mechanisms of drug resistance both in cell lines and primary murine T-ALLs. Cell lines are uniformly sensitive to GDC-0941, however gradual dose escalation results in resistant lines. In vivo, GDC-0941 is active against primary T-ALLs, but treated mice invariably relapse with drug resistant clones. Unexpectedly, a majority of GDC-0941-resistant cell lines and primary leukemias showed reduced levels of activated Notch1 protein, down-regulate many Notch1 target genes, and display cross-resistance to γ secretase inhibitors (GSIs). In multiple cases, Notch1 mutations that were present at enrollment were absent at relapse. Importantly, resistant clones that emerge both in vitro and in vivo up-regulate PI3K signaling indicating an “on pathway” mechanism of resistance. Consistent with these data, inhibition of Notch1 signaling promotes GDC-0941 resistance and enhances PI3K signaling, whereas expression of activated Notch1 increases the GDC-0941 sensitivity of mouse and human T-ALL cell lines. Thus, oncogenic Notch1 mutations that promote clonal outgrowth during malignant transformation unexpectedly undergo negative selection during treatment with GDC-0941. These studies validate PI3K as an important therapeutic target in T-ALL and raise the unexpected possibility that administering Notch1 and PI3K inhibitors in combination will accelerate drug resistance.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):PR08.

Citation Format: Monique Dail. Loss of oncogenic Notch1 signaling contributes to PI3 kinase inhibitor resistance in T lineage leukemia. [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 PR08.
Abstract 3960: MicroRNA-23b targeting urokinase-type plasminogen activator facilitates human papillomavirus type 16 E6 induction of cervical cancer cell migration
Authors: Yeung Chi Lam Au , Tsang Tsun Yee , Yau Pak Lun , Kwok Tim Tak
Published: August 2014

Aberrant expression of microRNA in human cervical cancer is associated frequently with human papillomavirus (HPV) integration. MicroRNA-23b (miR-23b) is often down-regulated in HPV-associated cervical cancer. Also, urokinase-type plasminogen activator (uPA), a target of miR-23b, is reported to be over-expressed in cervical cancer. Thus, in the present study, the functional role of miR-23b in HPV-16 E6 associated cervical cancer development was investigated.

Knockdown of oncoprotein E6 by E6 siRNA in HPV-16 positive human cervical carcinoma SiHa cells and CaSki cells led to an increase in the expression of miR-23b and a decrease in the expression of uPA as assessed by quantitative RT-PCR and Western blot analyses. Besides, uPA is confirmed to be the target of miR-23b as transient transfection of SiHa cells with miR-23b precursor vector repressed uPA expression and also the activity of luciferase reporter carrying the 3’UTR of uPA mRNA. uPA is known to be involved in cell migration and cell invasion. By wound healing assay, the cell migration rate of SiHa cells was reduced upon E6 as well as uPA knockdown. Moreover, the effect of E6 siRNA on cell migration was offset upon transfection with anti-miR-23b inhibitor. The results indicate that HPV-16 E6 may regulate cell migration through miR-23b/uPA pathway. Furthermore, p53 is a well-known gene target of HPV-16 E6. Knockdown of p53 by p53 siRNA decreased the miR-23b expression and increased the uPA expression. This shows that p53 may mediate the E6 effect on miR-23b/uPA expressions and thus cell migration of human cervical cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3960. doi:10.1158/1538-7445.AM2011-3960
Abstract LB-180: Epigenetic portraits of human breast cancers
Authors: Dedeurwaerder Sarah , Desmedt Christine , Calonne Emilie , Singhal Sandeep K. , Haibe-Kains Benjamin , Defrance Matthieu , Michiels Stefan , Volkmar Michael , Deplus Rachel , Luciani Judith , Lallemand Françoise , Larsimont Denis , Toussaint Jérôme , Haussy Sandy , Rothé Françoise , Rouas Ghizlane , Metzger Otto , Majjaj Samira , Saini Kamal , Putmans Pascale , Hames Gérald , van Baren Nicolas , Coulie Pierre G. , Piccart Martine , Sotiriou Christos , Fuks François
Published: August 2014

Background:

Understanding the diversity of breast cancer is essential to improving diagnosis and optimising treatment. Both genetic and acquired epigenetic abnormalities participate in cancer, but information is scant on the involvement of the epigenome in breast cancer and its contribution to the complexity of the disease. Our goal was to explore the DNA methylation landscapes of phenotypically heterogeneous tumours, to relate this diversity to landscape features, and extract biological and clinical meaningful information.

Methods:

We performed comprehensive DNA methylation profiling to assess the methylomes of two independent sets of frozen breast tissue samples: a “main set” of 123 samples (4 normal and 119 infiltrating ductal carcinomas, IDCs), and a “validation set” of 125 samples (8 normal and 117 IDCs). We used the recently developed Illumina's Infinium Methylation Assay, that allows to assess the methylation status of more than 27,000 CpGs corresponding to over 14,000 genes.

Results:

Firstly, it emerged that the two major phenotypes of breast cancers determined by ER status are widely epigenetically controlled. Secondly, we have distinguished, and validated in an independent set of tumours, 6 methylation-profile-based tumour groups, some coinciding with known “expression subtypes” but also new entities that may provide a meaningful basis for refining breast tumour taxonomy. Thirdly, we showed that DNA methylation profiling can reflect the cell type composition of the tumour microenvironment. Lastly, we highlighted an unexpectedly strong epigenetic component in the regulation of key immune pathways, revealing a set of immune genes having high prognostic value in specific tumour categories.

Conclusions:

In this study, we have generated the largest and most comprehensive DNA methylation data set for human breast tumor tissues. Several novel findings and original concepts for breast cancer emerge, that previous RNA expression profiling has not highlighted. By laying the ground for better understanding of breast cancer heterogeneity and improved tumor taxonomy, the precise epigenetic portraits drawn in our work should contribute to better management of breast cancer patients.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-180. doi:10.1158/1538-7445.AM2011-LB-180
Abstract LB-194: Phosphorylation-dependent dimerization of forkhead-associated domain regulates MDC1 function in response to DNA damage
Authors: Liu Jinping , Luo Shukun , Stern David , Ye Keqiong , Xu Xingzhi
Published: August 2014

MDC1 is a key mediator of DNA damage response in mammals. Here, we show that the N-terminal FHA domain of MDC1 mediates regulated dimerization in response to DNA damage. DNA damage induces phosphorylation of threonine 4 of MDC1 and FHA binds reciprocally to phospho-threonine-4 (pT4) from the other molecule. We determined the crystal structures of the MDC1 FHA domain in the free state and in complex with a pT4 peptide. FHA forms a dimer with pseudo 2-fold symmetry and face-to-face β-sheet contact. The FHA dimer is marginally stable, but is greatly stabilized in vitro and in vivo by the intermolecular binding between FHA and the preceding pT4 peptide. Forms of MDC1 with FHA deletion or mutations compromising dimerization are defective in foci formation at the sites of DNA damage, but could be largely rescued by an artificial dimerization module. Our results support a novel regulation mechanism of MDC1 function through T4 phosphorylation and FHA-mediated dimerization.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-194. doi:10.1158/1538-7445.AM2011-LB-194
Abstract LB-197: Hepatocyte Growth Factor Activator (HGFA) Inhibitors of c-MET/RON Kinase Signaling
Authors: Janetka James W. , Agarwal Manjula , Jones Darin E.
Published: August 2014

Continuous advances have been made in the treatment of cancer however 70% of the patients still die of cancer every year strongly correlated to tumor metastasis. The treatment options available at present are rarely able to effectively treat metastatic cancer. Targeting hepatocyte growth factor (HGF) signaling with receptor tyrosine kinase (RTK) inhibitors is a well-developed strategy for metastatic cancer therapy however no studies have explored targets upstream of kinase activation. One such target is hepatocyte growth factor activator (HGFA) which is upregulated in a large number of cancer cell lines and patient samples. HGFA is a trypsin-like serine protease which stimulates both the c-MET and RON signaling pathways by proteolysis of the inactive single-chain zymogen growth factor ligand precursors to activated two-chain HGF and macrophage stimulating protein (MSP) respectively. Increased activation of these pathways has been shown to trigger a number of downstream signaling events necessary for metastasis including cell migration, invasion, proliferation, and angiogenesis. The present study was undertaken to discover inhibitors of HGFA based on the naturally occurring bicyclic peptide, sunflower trypsin inhibitor (SFTI).

Using a combination of solution and solid-phase peptide methods, we have synthesized the natural product SFTI and analogs thereof. The compounds were tested for HGFA activity in a competitive fluorescence resonance energy transfer (FRET) assay of proteolytic cleavage by using a fluorogenic peptide substrate. We have identified several inhibitors of HGFA with low μM activity in the FRET assay. We have also demonstrated one inhibitor effectively prevented the conversion of native pro-HGF to HGF as determined by western blotting in a biochemical assay of HGFA activity. This ground breaking discovery of novel HGFA inhibitors will enable future studies on the role of HGFA and other serine proteases in the regulation of growth factors, cell signaling, and cancer metastasis. Inhibitors of HGFA offer an innovative therapeutic strategy upstream of kinase activation and signaling for the treatment of metastatic cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-197. doi:10.1158/1538-7445.AM2011-LB-197
Abstract LB-205: Using cell-delivered nanoparticles to cause local hyperthermia increases survival in a murine metastatic pancreatic cancer model
Authors: Basel Matthew T. , Balivada Sivasai , Wang Hongwang , Shrestha Tej B. , Seo Gwi Moon , Pyle Marla M. , Abayaweera Gayani , Dani Raj , Chikan Viktor , Bossmann Stefan , Troyer Deryl
Published: August 2014

Hyperthermia has been a method for cancer treatment for several decades now because cancer cells are slightly more susceptible to hyperthermia than healthy cells. Unfortunately, whole body hyperthermia has prohibitive side effects limiting its use or usefulness. Local hyperthermia, directed only to tumor tissue, could alleviate this problem and prove to be a potent cancer treatment. One method of generating local hyperthermia is to deliver magnetic nanoparticles to the tumor site and then generate heat using an alternating magnetic field (AMF). Here we demonstrate a system that uses tumor homing cells to actively carry iron/iron oxide nanoparticles into tumor tissue for AMF treatment. Paramagnetic iron/iron oxide nanoparticles were synthesized that absorb and convert AMF energy into heat very efficiently. These nanoparticles were loaded into Raw264.7 cells (mouse monocyte/macrophage like cells, Mo/Ma), that we have previously shown to be tumor homing cells. Test showed that the nanoparticles loaded at high concentration in the cells with very low toxicity. To test the system, a murine pancreatic cancer model was generated by injection of Pan02 cells i.p. After tumor development, Mo/Ma loaded with iron/iron oxide nanoparticles were injected i.p. and allowed to crawl into the tumor. Three days after injection, mice were exposed to an alternative magnetic field for twenty minutes to cause the cell-delivered nanoparticles to generate heat. This treatment regimen was repeated three times. A survival study demonstrated that this system can significantly increase survival in a murine pancreatic cancer model, with an average post-diagnosis life expectancy increase of 33%. Thus, for the first time, a cell-delivered nanoparticle system for generating localized hyperthermia has been demonstrated that can significantly prolong the life of i.p. pancreatic tumor bearing mice. This system has the potential to become a useful method for specifically and actively delivering nanoparticles for local hyperthermia treatment of cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-205. doi:10.1158/1538-7445.AM2011-LB-205
Abstract LB-306: Pre-treatment sera from NSCLC patients with different outcomes on EGFR-TKI therapy differentially affects sensitivity of lung cancer cell lines to EGFR-TKIs
Authors: Hunsucker Stephen W. , Grigorieva Julia , Helfrich Barbara A. , Allen Jenna , Bunn Paul A. , Roder Heinrich
Published: August 2014

Background: VeriStrat (VS) is a pre-treatment mass spectrometry-based test that correlates with clinical outcome after 2nd line therapy with gefitinib or erlotinib in advanced NSCLC. The blinded validation showed that patients classified as “Good” had significantly better overall survival than those classified as “Poor” (HR = 0.47 P = 0.0094 in one cohort, HR = 0.33 P = 0.0007 in another).(1) Mass spectra from VS Poor patients have relatively high abundance of eight peaks (proteins) and we believe these reflect an elevated host response to the tumor. Based on our clinical data and our understanding of the VeriStrat signature, we hypothesized that serum from VS Poor patients would increase the resistance of tumor cells to gefitinib in vitro.

Methods: The gefitinib sensitive line HCC4006 has an EGFR exon 19 deletion and the resistant line A549 is EGFR wild type. Human sera were from stage IIIB/IV NSCLC patients and characterized as VS Good or Poor. Pools were created by combining sera within each classification and used in growth inhibition assays. Cells were plated (10 replicates/drug concentration; 2,000 cells/well) using two media compositions; RPMI with 10% Good serum or RPMI with 10% Poor serum. After 24 hours, gefitinib was added and the plates were incubated for 6 days. The MTT assay was used to measure growth inhibition.

Conclusion: There was a relative decrease in inhibition of sensitive cells when grown in VS Poor serum, but no significant change in resistant tumor cells. We demonstrate that VS Poor serum has a different biological effect on tumor cells than VS Good serum. These results shed light on the nature of the host-tumor interaction and the relative efficacy of targeted therapies in patient populations. We will have similar data from 6–8 additional cell lines with and without EGFR mutations.

(1) Taguchi et al. J Natl Cancer Inst. 2007 99(11):838–46.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-306. doi:10.1158/1538-7445.AM2011-LB-306
Abstract LB-370: Radiation and hypoxia-induced bystander effect in human lung cancer cells
Authors: Tubin Slavisa , Gupta Seema , Ahmed Mansoor M.
Published: August 2014

Purpose: The bystander effects of radiation are well documented. However, it is shown that many cell lines that respire anaerobically do not show cytotoxic bystander effects. There is no existing data on the acute hypoxia-induced bystander effects. The purpose of this study was to investigate the status of radiation-induced bystander effect in normoxic, hypoxic and normoxic-hypoxic mixed conditions that are equivalent to in vivo tumor compartments as well as acute hypoxia-induced bystander effect. Methods: Two lung cancer cell lines, A549 and H460 were used. The cells were exposed either to real hypoxia (< 2 % oxygen) for 3 days or incubated in normoxic conditions and then irradiated (2 or 10Gy) after changing the media. After 24h (irradiated and unirradiated cells were continued to be in the hypoxic or normoxic environment respectively), unirradiated hypoxic (H-CM) or normoxic (N-CM) conditioned media and irradiated hypoxic (H-RCM) or normoxic (N-RCM) conditioned media was collected and cells were incubated with fresh media for another 24h before collecting the second respective CM. Unirradiated normoxic cells were exposed to H-CM, N-CM, H-RCM or N-RCM and cell growth was monitored dynamically in real time using real time cell electronic sensing system (RT-CES). Results: Our preliminary results in A549 cells showed that H-RCM was much more effective in inducing growth delay compared to N-RCM. For all the treatments except for 2Gy N-RCM, which promoted slight cellular growth, RCM induced significant growth delay compared to CM. Further, as expected N-RCM obtained after 10Gy irradiation was more cytotoxic/cytostatic than 2Gy N-RCM.

Interestingly, H-RCM obtained after 2Gy and 10Gy irradiation did not show significant differences in the growth delay of A549 cells. During the first 48h under real hypoxic conditions (24 or 48h H-CM), the H-CM promoted slight increase in cellular growth followed by significant growth delay which were most pronounced at 96h. When, in addition to the bystander factors, the cells were exposed to 2Gy direct irradiation, the growth was severely affected. Currently, we are performing these experiments in more radiosensitive cell line, H460 and also investigating the effects of acute hypoxia and mixed normoxia-hypoxia on generation of bystander effects. Conclusions: The findings from this study demonstrate for the first time that irradiation of hypoxic cells and exposing the cells to acute hypoxia lead to significant bystander effect that has important clinical implications.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-370. doi:10.1158/1538-7445.AM2011-LB-370
Abstract LB-374: IGFBP-6, a novel prognostic biomarker in nasopharyngeal carcinoma (NPC) patients
Authors: Lu Yi , Chen Qiuyan , Qi Weici , Hong Minghuang , Mizokami Atsushi , Keller Evan , Zhang Jian
Published: August 2014

A variety of cancer types, including NPC, that have metastasized to bone, have a dysregulated insulin-like growth factor (IGF) system. This suggests that IGF-IGF receptor axis may contribute to the interaction between host and cancer cells resulting in promotion of skeletal metastases. In this study, IGFBP6 expression levels were first determined by immunohistochemistry in clinical specimens from NPC patients (n=76). IGFBP6 was differentially expressed in malignant epithelia and in non-neoplastic tissues. The 5-year overall survival rate of IGFBP-6 negative and positive group was 27% and 73% (survival analysis P=0.007), respectively and the 5-year distant metastasis-free survival rate was 53% and 89% (P=0.002), respectively. Multivariate analysis in a Cox model showed that IGFBP-6 was an independent and positive prognostic biomarker in NPC patients. Furthermore, we found that in vitro cell proliferation and invasion were inhibited by recombinant IGFBP6 treatment in CNE2 cells but not in HK-1 cells. Finally, CNE2 cells were stably transfected with IGFBP6 short hairpin RNA (shRNA) or scrambled RNA. The cells were inoculated into the tibiae of SCID mice for 3 weeks. The IGFBP6 knockdown significantly enhanced CNE2 tumor growth in bone. These results indicate that IGFBP6 may play key roles in NPC tumor progression. We conclude that, for the first time, IGFBP-6 is a novel prognostic biomarker in NPC patients.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-374. doi:10.1158/1538-7445.AM2011-LB-374
Abstract LB-4: Mechanisms by which the unfolded protein response/α-Basic-crystallin (CRYAB) regulates VEGF signaling of tumor endothelial cells
Authors: Cai Jun , Ruan Qing , Han Song , Chen Zhijuan , Law Brian K. , Jiang Wengou
Published: August 2014

Angiogenesis is an essential component of tumour development and is tightly regulated by a network of pro-angiogenic and anti-angiogenic factors. The tumour cells are known to produce many of the proteins that interact with vascular endothelial cells and drive the angiogenic process. Of the known angiogenic factors, VEGF has been established as a potent inducer of tumor angiogenesis. Like almost all secretory and membrane proteins, VEGF attains its final folded or assembly conformation in ER before making its way to its target organelles or the cell surface. The deleterious tumor microenvironment characterized by progressive hypoxia, nutrient starvation and acidosis results in the ER stress in the tumor vascular endothelial cells, which in turn leads to the unfolded/misfolded VEGF accumulated in the ER.

Molecular chaperon proteins counteract the formation of aberrantly folded VEGF and allow its correct folding. One of molecular chaperons is CRYAB. It has been reported that CRYAB contains interactive sequences for VEGF. The misfolded VEGF interacts with BiP (chaperone immunoglobulin heavy chain-binding protein) causing its release from one or more of the three ER stress sensors [PKR-like ER kinase (PERK), inositol-requiring protein-1 (IRE1) and transcription factor 6 (ATF6)], which leads to activation of the unfolded protein response (UPR) pathway. All three sensor pathways can lead to chaperone induction but the IRE1 and ATF6 pathways are thought to play the major role in up-regulation of CRYAB. Activation of IRE1 generates a mature XBP-1 mRNA by unconventional splicing of XBP-1 pre-mRNA and activated XBP-1 up-regulates CRYAB. Activation of ATF6 results in its translocation to the Golgi apparatus and proteolytic cleavage to yield its cytosolic domain not only for directly up-regulating genes encoding CRYAB and folding catalysts, but also for assisting IRE1-dependent upregulation of CRYAB. An excessively elevated level of CRYAB has been detected in breast carcinomas, correlating with lymph node involvement resulting in metastasis and short term survival. Taken together, it is clear that CRYAB has the potential to a) protect VEGF from intracellular degradation thus maintaining the intracellular pool and b) increase VEGF secretion. Both these actions serve to enhance the autocrine VEGF signaling response which serves as one of strategies adapted by the tumor vasculature to perpetuate aberrant angiogenesis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-4. doi:10.1158/1538-7445.AM2011-LB-4
Abstract 1006: A novel class I histone deacetylase (HDAC) inhibitor, paragazole, demonstrates antiproliferative and proapoptotic effects in triple-negative breast cancer models.
Authors: Astling David P. , Tentler John J. , Cross Benjamin R. , Newton Timothy P. , Pitts Todd M. , Gunther Eric , Liu Xuedong , Eckhardt S. Gail , Diamond Jennifer R.
Published: August 2014

Introduction: Triple-negative breast cancer (TNBC) is a clinically aggressive breast cancer subtype which lacks estrogen receptor expression. Histone deacetylase (HDAC) inhibitors have previously been shown to induce estrogen receptor expression in TNBC models. Paragazole is a selective class I HDAC inhibitor (HDACs 1, 2, and 3) currently in preclinical development. In this project, we explored the antiproliferative and proapoptotic activity of paragazole and investigated the combination of paragazole and chemotherapy in a panel of breast cancer cell lines.

Methods: Breast cancer cell lines were exposed to varying sub-micromolar concentrations of paragazole alone and in combination with carboplatin, paclitaxel, and gemcitabine. Proliferation was assessed using an SRB assay and analyzed using the Calcusyn program, whereby synergy was defined as a Combination Index of less than 1. Cell cycle analysis was performed using flow cytometry and apoptosis was analyzed using a caspase 3/7 assay. Baseline HDAC expression was measured by RT-PCR and RNA-Seq was used pre- and post-treatment in a subset of 2 sensitive and 2 resistant cell lines.

Results: Exposure to paragazole resulted in a decrease in cell proliferation at submicromolar concentrations with more robust antiproliferative activity observed in the TNBC cell lines compared to the estrogen receptor positive cell lines. An increase in apoptosis that was maximal at 24-48 hours was observed with single agent paragazole in a subset of sensitive TNBC cell lines. The combinations of paragazole with either paclitaxel, gemcitabine, or carboplatin all resulted in additive or synergistic growth inhibition. Expression of HDAC 1 was higher in the sensitive TNBC cell lines than in the resistant lines. Exposure to paragazole led to an increase in CARM1 mediated estrogen receptor expression.

Discussion: These in vitro results demonstrate that paragazole has antiproliferative and proapototic activity against TNBC cell lines and may potentiate the activity of chemotherapy. These data support confirmation of the findings using in vivo models and the investigation of paragazole in combination with anti-estrogen agents such as tamoxifen and fulvestrant.

Citation Format: David P. Astling, John J. Tentler, Benjamin R. Cross, Timothy P. Newton, Todd M. Pitts, Eric Gunther, Xuedong Liu, S. Gail Eckhardt, Jennifer R. Diamond. A novel class I histone deacetylase (HDAC) inhibitor, paragazole, demonstrates antiproliferative and proapoptotic effects in triple-negative breast cancer models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1006. doi:10.1158/1538-7445.AM2013-1006