bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3253: Novel oxindole inhibitors of Aurora A kinase: Structure based hit-to-lead approach
Authors: Pireddu Roberta , Lawrence Harshani R. , Martin Mathew P. , Betzi Stephane , Yip Richard , Yang Hua , Sun Nan N. , Cheng Jin Q. , Schonbrunn Ernst , Sebti Said M. , Lawrence Nicholas J.
Published: June 2012

The M-phase of cell division is controlled by phosphorylation events performed by several mitotic serine/threonine kinases. For example, the three human isoforms of Aurora kinase (A, B, and C) have been amply investigated as they are essential for the execution of numerous mitotic events and required for genome integrity and stability. Studies have shown that Aurora A and B are frequently over-expressed in various cancer cells. Aurora A also acts as an oncogene inducing malignant transformation when over-expressed. Since the discovery of the Aurora kinases and their association with cancer, there has been great interest in developing small molecule inhibitors of Aurora kinases as potential anticancer drugs.In-house screening campaign directed toward the discovery of novel Aurora A inhibitors, led to the identification of a new and potent class of oxindole derivatives as inhibitors of Aurora-A kinase. The initial hit HL1-058-1 was identified as an inhibitor of histone H3-Ser10 phosphorylation (IC50 1-5 μM).

By using a Z’-lyte kinase assay kit, the inhibitory effect of HL1-058-1 was shown to be the direct consequence of the Aurora kinase inhibition (IC50 of 2.7 ± 0.3 µM). Based on the preliminary data, HL1-058-1 was selected for further study. Synthesis of a focused library of analogs based on this hit revealed RPM305 and RPM304 as potent inhibitors of Aurora A (IC50 0.020 ± 0.005 μM, and 0.038 ± 0.018 μM, respectively). Interestingly, compound RPM304 showed 26 fold selectivity over Aurora B (IC50 0.99 ± 0.08 μM).

A coupled enzyme assay was also used to carry out further structure activity relationship studies of the most potent analogs.

One of the lead compounds was co-crystallized with the kinase domain of Aurora A. The design and synthesis of further focused libraries of oxindoles, based on this X-ray structure will be described. Details of the cell activity of this class of compound to inhibit Aurora A activity and tumor cell growth will be described.

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 3253. doi:10.1158/1538-7445.AM2011-3253
Abstract 3255: Tetrahydropyridine derivatives: Novel and potent histone demethylase LSD1 inhibitors
Authors: Callery Patrick S. , Wichitnithad Wisut , Borysiewicz Elizabeth , Konat Gregory W. , Konat Gregory W.
Published: June 2012

Flavin-dependent histone demethylases play an important role in epigenetic gene regulation. The most studied enzyme, LSD1 (KDM1), catalyzes the oxidative demethylation of methylated Lys4 of histone H3. Known LSD1 inhibitors also inhibit other flavin-dependent enzymes, in particular monoamine oxidase (MAO). Therefore, selective inhibition of LSD1 is a significant drug discovery target. We designed, synthesized and evaluated in vitro a series of eleven N-substituted tetrahydropyridines as inhibitors of flavin-dependent enzymes. Complete chemical structures of the compounds used will be disclosed at the time of presentation. The inhibition of LSD1 was measured in rat glioma C6 cells. Briefly, cells grown in serum-free medium were treated with the inhibitors for 24 h, and the level of Lys4 dimethylated form of histone H3 was measured by immunoblot. MAO A activity was determined using human recombinant enzyme with kynuramine as substrate. The most selective among the inhibitors showed low nanomolar potency toward LSD1 and weak inhibition of MAO A. The observed high potency and substrate selectivity justifies further evaluation of the drug discovery potential of the tetrahydropyridine scaffold.

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 3255. doi:10.1158/1538-7445.AM2011-3255
Abstract 3260: Identification and synthesis of benzimidazole carboxamides as potent inhibitors of focal adhesion kinase (FAK)
Authors: Verma Sharad K. , Leber Jack D. , LaFrance Louis V. , Li Mei , Duquenne Celine , Cui Guanglei , Miller William H.
Published: June 2012

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that integrates signals from integrins and growth factor receptors. FAK has been reported to play a role in the regulation of cell survival, growth, spreading, migration and invasion. Overexpression of FAK mRNA and/or protein has been documented in many human tumors, including cancers of the breast, colon, thyroid, and prostate. FAK is regulated and activated by phosphorylation on multiple tyrosine residues. More significantly, there is evidence that phosphorylated FAK is increased in malignant compared to normal tissues. Accordingly, compounds that inhibit FAK activity are expected to be useful for the treatment of cancer. Cross-screening of the GSK compound collection led to the identification of benzimidazole carboxamides having weak FAK inhibitory activity. As benzimidazole carboxamides are known kinase templates, a structure based hit-to-lead optimization campaign was executed. Team efforts culminated in the rapid identification of a series of substituted benzimidazole carboxamides having highly potent and selective inhibitory activity towards FAK (i.e. FAK IC50 < 10 nM). The structure-based optimization of this series including the synthesis, binding mode, and profile of lead compounds will be described.

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 3260. doi:10.1158/1538-7445.AM2011-3260
Abstract 3283: VB-111 a novel anti-angiogenic vector and a promising treatment for metastatic cancer in combination with other anticancer drugs
Authors: Breitbart Eyal , Cohen Yael C. , Bangio Livnat
Published: June 2012

Background: VB-111 is a vascular disruptive and anti-angiogenic agent consisting of a non-replicating adenovirus vector (Ad-5, E1 deleted), which contains a novel Vascular Targeting System™, VTS™, and a fas and human tumor necrosis factor receptor chimeric transgene. This modified promoter specifically expresses the fas chimera transgene in tumor vessel endothelium, leading to targeted apoptosis. Preclinical in vitro and in vivo studies as well as a phase I clinical trial, have shown that VB-111 was safe and well tolerated, and had significant antitumor activity. We report here the results of a comprehensive set of in vivo studies of VB-111 in combination with chemotherapeutic and anti-angiogenic drugs.

Methods: The safety and efficacy of VB-111 were evaluated employing the Lewis Lung metastasis model as a monotherapy, in combination with bevacizumab and in combination with Paclitaxel.

Results: VB-111 was found to induce a dose dependent reduction of up to 90% in tumor burden of lung metastases in Lewis Lung metastasis model. Evaluation of the combined therapy with VB-111 and Avastin in Lewis Lung metastasis model showed no additive effect of Avastin 4 or 8 mg/kg as compared to VB-111 as a monotherapy. Additive effect was seen when mice were treated with a lower dose of VB-111 (1×10E9 VP/mouse) in combination with Paclitaxel (40mg/kg) and with higher dose of VB-111 (1×10E11 VP/mouse) with Paclitaxel (20mg/kg). No toxicity was observed as a result of the combined therapy, except for the lungs the organ weights among the different treatment groups showed minimal variation. In all the mice tested, the harvested organs were found to have normal growth pathology with no lesions or evidence of metastasis.

Monotherapy with VB-111 did not result in any toxicity and combination therapy did not introduce additional toxicities.

Conclusions: As previously shown, VB-111 was found to be safe and specific. VB-111 may provide additive effect to chemotherapy in a combination regimen. This will be further explored in clinical trials.

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 3283. doi:10.1158/1538-7445.AM2011-3283
Abstract 3310: Regulation of reprogramming factors by c-Met signaling in glioblastoma stem cells
Authors: Li Yunqing , Li Angela , Ying Mingyao , John Laterra
Published: June 2012

Reprogramming transcription factors such as Sox2, c-Myc, Klf4, Oct4 and Nanog have an essential role in sustaining the growth and self-renewal of embryonic stem cells. Forced expression of these reprogramming factors can reprogram differentiated somatic cells into pluripotent stem cells. Reprogramming factor overexpression has also been found in human cancer stem-like cells including glioblastoma stem cells (GBM SCs). This suggests that reprogramming factors support the self-renewing and tumor-initiating phenotype of cancer stem cells. Hence, the molecular signaling pathways that regulate reprogramming factor and their support of the cancer stem-like phenotype in GBM and other solid tumors are of considerable interest. We recently found that the tyrosine kinase c-Met oncogene up-regulates a network of reprogramming factors consisting of Oct4. Sox2, Klf4, c-Myc and Nanog in GBM-derived neurospheres that are enriched in GBM SCs. Reprogramming factor induction by c-Met was observed under serum-free culture conditions that support neurosphere-formation and in cells that were pre-differentiated in serum-containing medium. Using flow cytometry and quantitative reverse transcription-PCR, we also found that GBM neurosphere cell subpopulations expressing c-Met at high levels also express high levels of Nanog and Sox2 in comparison to low c-Met expressing cells. Using specific pharmacological inhibitors of PI(3)kinase (LY294002, wortmannin), MAPkinase (PD98059, U0126), and Stat3 (Stattic, Janus Kinase JSI-124), we found that c-Met signaling regulates reprogramming factors mainly via two parallel pathways: the Jak-Stat3 and PI(3)K-Akt pathways. Inhibiting Oct4 induction abrogated the capacity of c-Met to induce Sox2, c-Myc, and Nanog but not KLF4 expression. Inhibiting Nanog induction significantly inhibited (84% inhibition) the capacity of c-Met signaling to stimulate GBM SC self renewal as measured by neurosphere formation and Edu incorporation, 84% and 61% inhibition, respectively. Together, these findings indicate that c-Met signaling upregulates an Oct4- and Nanog-dependent reprogramming factor network that supports GBM SCs.

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 3310. doi:10.1158/1538-7445.AM2011-3310
Abstract 3321: The benefits from combined temozolomide and sunitinib in medulloblastoma cells: The antitumor effect, inhibition of invasiveness, and blocking of cancer stem cells
Authors: Kim Dong-Ok , Ra Young Shin
Published: June 2012

Objectives: Medulloblastoma is the most common malignant and metastatic pediatric brain tumor in children. Angiogenesis inhibitors, such as sunitinib, represent a promising strategy to improve glioblastoma (GBM) tumor response. Temozolomide (TMZ) also became the standard of care for GBM patients by removing alkyl groups from the O6-position of guanine. But, there is little evidence about migration and invasion study on medulloblastoma cells treated with sunitinib or TMZ. In this study, we identified the effects of sunitinib or TMZ only, and the combination of them on migration and invasiveness in medulloblastoma cell line (DAOY). Also the characteristics of cancer stem cells were investigated. Methods and Results: The cell proliferation rate showed TMZ (1000µM) 70%, sunitinib (62.5µM) 60%, and the combination of TMZ and sunitinib (1000/62.5µM) 35% at 72hr after treatment with the drugs in DAOY cells. Treatment with sunitinib in addition to TMZ in the cells demonstrated more G2/M arrest than cells treated respective drugs at 24hr exposure of the drugs from cell cycle analysis. Through Rhodamine phalloidin staining, we also observed change of cell size and F-actin alterations in all cells treated TMZ, sunitinib only or the combination of the reagents, in contrast to cells treated DMSO as a control. And, in invasion assay using Matrigel chamber, the cells treated with combination of drugs were inhibited invasiveness the most when compared with control, sunitinib, and TMZ only at 72hr after exposure to the drugs. Moreover, when the cells were treated with sunitinib and TMZ simultaneously, migration ability from wound-healing assay represented higher than in cells treated TMZ or sunitinib only during exposure of 24hr. We investigated properties of cancer stem cells as well as characteristics of migration and invasion in cells treated the drugs analyzing the expression of protein during exposure of 24, 48, 72hr by western blotting. As a result, we identified the decreased levels of the protein such as p-PDGFR-β, p-VEGFR1, integrin-β1, MMP-9, CD44, Rac1 as migration and invasion markers, nestin, CD133, Oct3/4, Sox2 as cancer stem cell markers and O6-methylguanine methyltransferase(MGMT). Conclusions: Taken together, the combination therapy functions as very effective therapeutic tool in terms of, not only inhibititor of proliferation, migration and invasiveness, but also cancer stem cells in medulloblastoma cell line.

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 3321. doi:10.1158/1538-7445.AM2011-3321
Abstract 3359: Maspin induces MET in prostate cancer cells cultured in 3D-collagen I
Authors: Dean Ivory , Bernardo Margarida , Li Xiaohua , Dzinic Sijana , Yin Shuping , Moin Kamir , Sakr Wael , Sheng Shijie
Published: June 2012

The aggressiveness of carcinoma is typically associated with a significant epithelial-to-mesenchymal phenotypical shift (EMT). While several pathways have been identified as the driving force for EMT, mechanisms that epithelial cells depend on to prevent or reverse EMT are largely unknown. To this end, the tumor suppressive maspin inhibits tumor cell motility, invasion, pericellular plasminogen activation and extracellular matrix (ECM) degradation, and blocks the prostate tumor-induced bone remodeling. Furthermore, a general correlation between maspin expression and better differentiated epithelial phenotypes has been noted, with established cell lines, in an in vivo model for prostate tumor bone metastasis and in human prostate tissue specimens. The objective of the current study is to test whether maspin is sufficient to drive the program of mesenchymal-to-epithelial transition (MET). We showed that stably transfected DU145 that express maspin (M7) formed spheroid structures of polarized cells with a hollow lumen in 3D collagen I culture. The M7 acini in 3D collagen were encircled with reorganized fibrillar collagen, while the Neo control cells degraded collagen fibrils. The spheroid formation was abolished when M7 cells were transfected with maspin shRNA. Under the same condition, no redifferentiation of DU145 cells, with or without maspin, occurred in 3D Matrigel. To examine what other cellular and molecular changes are associated with the maspin-led tumor cell redifferentiation, we performed immunofluorescent staining and found that maspin correlated with highly organized β1-integrin localized on the periphery of the M7 spheroids. We confirmed the secretion of maspin protein by prostate epithelial cells. To test whether the effect of maspin on prostate epithelial cell differentiation depends on uPA or uPAR, we performed the 3D collagen spheroid formation assay in the presence of specific neutralizing antibodies. We found that only the neutralizing antibodies of either uPA or uPAR led to a slight increase of the size of the spheroids. While uPA and uPAR may be targeted as a part of the biological activities of maspin, our results suggest that the effect of maspin more than just blocks the uPA/uPAR cascade. Indeed, mRNA microarray, with cDNA derived from Neo and M7 cells grown in 2D cultures, 3D culture in collagen I and bone tumors in the Scid-Hu model, identified 32 commonly up-regulated genes and commonly down-regulated genes upon maspin expression. These epigenetic changes, along with other evidence, support the hypothesis that maspin induces MET in prostate tumor cells.

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 3359. doi:10.1158/1538-7445.AM2011-3359
Abstract 3361: Phosphoglucose isomerase/autocrine motility factor mediates epithelial-mesenchymal transition regulated by miR-200 in breast cancer cells
Authors: Ahmad Aamir , Aboukameel Amro , Kong Dejuan , Wang Zhiwei , Raz Avraham , Sarkar Fazlul H.
Published: June 2012

Phosphoglucose isomerase/autocrine motility factor (PGI/AMF), a housekeeping gene product that plays a key role in both glycolysis and gluconeogenesis pathways, is associated with invasion and metastasis of cancer cells. PGI/AMF is involved in the induction of Epithelial-to-Mesenchymal transition (EMT) phenotype in breast cancer cells, which leads to increased aggressiveness; however, the molecular mechanism by which PGI/AMF regulates EMT is not known. Here, we show, for the first time, that the over-expression of PGI/AMF leads to an increase in the DNA-binding activity of NF-κB, which in turn causes increased expression of ZEB1/ZEB2. Since microRNA-200s (miR-200s; miR-200a, miR-200b and miR-200c) are known to negatively regulate the expression of ZEB1/ZEB2, we assessed their expression and found that the expression of miR-200s was lost in PGI/AMF over-expressing MCF-10A cells as well as in highly invasive MDA-MB-231 cells, which was consistent with increased expression of ZEB1/ZEB2. Moreover, silencing of PGI/AMF expression in MDA-MB-231 cells led to re-expression of miR-200s, which was associated with the reversal of EMT phenotype i.e. Mesenchymal-to-Epithelial Transition (MET), and these findings were consistent with alterations in the relative expression of epithelial (E-cadherin) and mesenchymal (vimentin, ZEB1, ZEB2) markers, and decreased aggressiveness, as judged by clonogenic, motility and invasion assays. Silencing of PGI/AMF was also found to result in significantly reduced pulmonary metastases in vivo. Collectively, these results suggest, for the first time, the role of miR-200s in PGI/AMF induced EMT-MET, and thus clinical approaches for up-regulation of miR-200s could become a novel therapeutic strategy for the treatment of highly invasive breast 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 3361. doi:10.1158/1538-7445.AM2011-3361
Abstract 337: HPV16 infection plays a significant role in laryngeal carcinogenesis among Puerto Ricans
Authors: Vélez-Reyes Germán L. , Ruiz-Fullana Francisco J. , García Aurian , Trinidad-Pinedo Juan , Baez Adriana
Published: June 2012

Aims: Incidence of head and neck cancer (HNSCC) has increased in the past decades in younger adults. Oncogenic proteins E6 and E7 in HPV 16 are known to block the p53 and pRB pathways leading to abnormal cell growth and development of cancer. We have shown that human papillomavirus type 16 (HPV-16) plays a significant role in the etiology of head and neck cancer in the Puerto Rican population. Furthermore, there is an increasing trend in the incidence of oropharyngeal cancers, particularly among men under 50 years old, for which HPV infection is the likely cause. This study aims to evaluate the association between clinicopathologic and demographic characteristics of HPV16- positive HNSCC Puerto Rican patients.

Methodology: Clinical and pathological data of all newly diagnosed cancer patients was collected, after signing the informed consent. The distribution by anatomical site was as follows: 86 larynx, 16 oropharynx, 12 hypopharynx, and 70 in the oral cavity. Detection and quantification of HPV16 E6 oncoproteins in tumor tissue was made by real time PCR technology. Associations between HPV status, risk factors and tumor characteristics were analyzed.

Results: HPV16 was found in 52% of tumors tested. When the HNSCCs were analyzed by anatomical site for HPV-status, 52% of the larynx, 53% of oral cavity, 62% of oropharynx, and 42% of the hypopharynx were HPV-positive. The highest prevalence was observed in patients under the age of 60 years. No significant association was found between HPV status and histological grade, alcohol usage, and smoking status.

Conclusions: Our results suggest that the high HPV DNA present in laryngeal HNSCCs indicates that HPV16 has significant role in the development of SCC of the larynx in Puerto Ricans. These results may be further validated in a larger sample of 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 337. doi:10.1158/1538-7445.AM2011-337
Abstract 3375: The epithelial to endotheliod transition (EET): A cellular modulation that promotes the head and neck squamous cell carcinoma tumorigenic phenotype
Authors: Tong Meng , He Lingli , Han Byungdo , Holpuch Andrew , Pei Ping , Mallery Susan R.
Published: June 2012

The presence of the EMT (epithelial-mesenchymal transition) and EndMT (endothelial-mesenchymal transition) demonstrates the extent of phenotypic plasticity in cancers. Previous findings in head and neck squamous cell carcinoma (HNSCC) cells, i.e., VEGF's autocrine-paracrine function and their responsiveness to endostatin, imply that HNSCC cells share some functional characteristics with endothelial cells and suggest the potential for an “epithelial-to-endotheliod transition (EET)”. Such a transition would facilitate HNSCC tumorigenesis by augmenting release of proangiogenic factors and facilitating tumor cell migration and invasion. Consequently, these studies elucidated whether or not an EET occurs in HNSCC cells and tumors, and evaluated cellular parameters to uniquely characterize this epithelial-endotheliod transition. Our results demonstrate that cultured HNSCC cells express a variety of endothelial-specific markers, and assumption of the endotheliod phenotype has functional consequences including uptake of acetylated low density lipoprotein and enhanced invasion and migration. Selective HNSCC cells were also highly responsive to transforming growth factor-β1 and express its auxiliary receptor, endoglin. Furthermore, the EET is not restricted to cultured HNSCC cells per se as tumor cells in resected human HNSCC tumors and proliferating epithelia at the margins of ulcers demonstrate a similar phenotype. These data suggest that, similar to EMT, the EET enhances epithelial cell migration. Also, the regulation of this transition-transient (wound healing) versus sustained (invasive cancers)-impacts clinical outcome. Finally, recognition of the EET as a distinct entity provides impetus for future clinical trials to concurrently assess the antitumorigenic as well as the angiostatic properties of therapeutic agents formally regarded as exclusively angiostatic in scope.

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 3375. doi:10.1158/1538-7445.AM2011-3375
Abstract 3384: The effects of mono- and combination-treatment of phytochemicals and antimicrotubule chemotherapeutic agents on prostate cancer cell proliferation and expression of EMT markers
Authors: Smith Basil A.
Published: June 2012

PURPOSE: Treatment of androgen-independent metastatic prostate cancer with chemotherapeutic agent Estramustine showed diminishing success. Epidemiological studies showed an association between reduced cancer risk and dietary intake rich in phytochemicals from fruits and vegetables. We studied the molecular mechanisms through which phytochemicals muscadine grape skin extract (MSKE) and camalexin act individually, or in combination with Estramustine, to affect epithelial-mesenchymal transition (EMT), a process involved in prostate cancer progression. EMT can be induced by transcription factors such as Snail, and is associated with decrease in cell adhesion molecules like E-cadherin, and increase in mesenchymal markers such as vimentin. METHODOLOGY: MSKE, Camalexin, and Estramustine, were used in varying concentrations individually, and in combination, to conduct MTS proliferation assay on C4-2 and ARCaP prostate cancer EMT model overexpressing constitutively active Snail cDNA. EMT markers, E-cadherin and vimentin, were examined by Western Blot Analysis following treatments. RESULTS: MSKE or Camalexin with or without Estramustine significantly decreased cell proliferation of C4-2 cells. Surprisingly, ARCaP-Snail cells were more sensitive to MSKE and Estramustine treatments, and showed significant decrease in viability, as compared to control ARCaP-Neo cells. ARCaP-Snail cells treated with 5µg/ml MSKE showed a reversal in EMT as shown by increased E-cadherin and decreased vimentin levels. CONCLUSION: Both mono and combination treatments retarded prostate cancer cell proliferation and may show promise as a therapeutic agent to abrogate EMT and prostate cancer metastasis in the future. Supported by grants P20MD002285-01 & G12RR003062-22.

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 3384. doi:10.1158/1538-7445.AM2011-3384
Abstract 3394: A gamma secretase inhibitor reduces large neurosphere formation in glioma cell lines which correlates with decreased tumorigenicity
Authors: Lopez-Molina Javier , Rao Sudhir , Angagaw Minilik , Wexler Roseanne , Cristescu Razvan , Demuth Tim , Sun Hong , Toniatti Carlo
Published: June 2012

Notch signaling regulates many aspects of development, cell fate and tissue renewal. Recent work has implicated the pathway in regulating and maintaining tumor initiating cells. Phase 1 clinical trial results have recently shown responses in patients with brain tumors. The objective of the current study is to evaluate the effect of a potent and specific gamma-secretase inhibitor (GSI, MRK-003) on the growth of a neurosphere stem cell-like population from glioblastoma cell lines. Our studies demonstrate glioma cell lines grown in serum-free neurosphere culture conditions have 10-40 fold higher expression of Notch pathway direct target genes Hes5, Hey1 and Hey 2, as compared to their expression in standard 2D serum-cultured cells. Using a novel imaging based readout with the Isocyte platform, we have collected multiple neurosphere colony parameters, such as colony size, colony number, colony shape, frequency and fluorescence intensity, before and after drug treatment. In vitro GSI treatment of U251 neurospheres as compared to DMSO and inactive enantiomer controls yields a 5 fold reduction in the number of large colonies with a correlated increase in the number of small colonies, suggesting a cytostatic effect. This effect translates into a 40 day delay in tumor formation and a 5 fold decrease in tumor volume after subcutaneous injection of 5-day GSI pre-treated neurospheres (p-value<0.0001, 1way-ANOVA). This data suggests that GSI inhibits the growth of tumor initiating cells that are present in neurosphere culture and that this change is specifically mediated by the Notch pathway. Furthermore, this imaging based assay will allow for the validation of Notch pathway related gene signatures for their ability to predict response to GSI in this cancer context.

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 3394. doi:10.1158/1538-7445.AM2011-3394
Abstract 34: Microenvironmental independence or co-option: How tumour-stromal interactions influence prostate cancer lethal outcomes
Authors: Basanta David , Hashim Arig Ibrahim , Gillies Robert J. , Gatenby Robert A. , Ayala Gustavo E. , Hayward Simon W. , Anderson Alexander RA
Published: June 2012

The role of the tumour-microenvironment interactions in tumor progression has been the extensively studied in the last few years. Recently, the key role that stromal interactions in both cancer initiation and progression has become an important focus. Both of these interactions, cell-cell and cell-microenvironment, can fundamentally affect the outcome of tumour progression. In prostate cancer this is particularly true, with the tumour microenvironment being an independent predictor of patient prognosis. There are two main lethal outcomes for prostate cancer:

(i) early in its genesis the tumour acquires a highly malignant lethal phenotype, growing rapidly and displacing the original stromal population. These less common aggressive tumours are independent of much of the local microenvironment.

(ii) the tumour co-opts the local stroma taking on a classic stromagenic phenotype where the local microenvironment facilitates cancer growth.

Using an integrated approach we have investigated the influence of tumour stroma interactions in driving these outcomes. With the help of a mathematical model based on evolutionary game theory, we considered three distinct cellular populations: stromal cells, tumour cells that are self-reliant in terms of microenvironmental factors and tumour cells that depend on the environment for resources (such as growth factors, nutrients and space) but can also co-opt stroma. The results from this mathematical model highlight the importance of carcinoma associated fibroblasts in stromagenic outcomes and that the resulting cooperation between certain stromal cells and the tumour could be disrupted as a result of changes in the microenvironment leading to a stromal independent outcome. Subsequently we have employed a biological model using TRAMP mice (TRansgenic Adenocarcinoma of the Mouse Prostate). TRAMP mice develop prostate cancer spontaneously after a few weeks and recapitulate many features of prostate cancer in humans. This mouse model allowed us to investigate the differences between the tumour populations driving the two distinct outcomes as well as the importance of microenvironmental changes in diverting this progression.

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 34. doi:10.1158/1538-7445.AM2011-34
Abstract 340: Predictive value of KRAS mutations in advanced non-small cell lung cancer (NSCLC) patients (pts) treated with chemotherapy (CT)
Authors: Dingemans Anne-Marie C. , Smit Egbert F. , Derks Jules , Mellema Wouter , Heideman Danielle , Van Suylen Robertjan , Thunnissen Erik
Published: June 2012

Introduction: KRAS mutation is thought to be related with poor outcome of NSCLC pts. The predictive value of KRAS mutation for response to chemotherapy (CT) has not been established

Patients and methods: From a retrospective database from 2 university hospitals all pts with advanced NSCLC treated with palliative platinum containing chemotherapy were selected. Pts were included when archive tumor tissue was available for DNA extraction. High resolution melting followed by PCR sequencing was performed for KRAS exon1 and 2.

Results: KRAS mutation analysis was evaluable for 122 pts. All pts had advanced NSCLC and were no candidate for treatment with curative intent. Mean age 61 years (range 24-80 years), 81 males and 41 females. All pts received platinum-containing CT. Response was evaluable for 110 pts: 1 complete response, 21 partial response, 54 stable disease and 54 progressive disease. Median progression free survival (PFS) was 5.7 months (m) (95% CI 4.2-7.2 m), median overall survival (OS) was 7.5 m (94% CI 5.2-9.9 m). 44 pts (36%) had a KRAS mutation. 38% of pts with a KRAS mutation had as best response PD on CT, and 27% of pts without a KRAS mutation had PD, this difference was not significant. No difference in PFS and OS was observed for pts with a KRAS mutation (median PFS 5.3 m, 95% CI 3.2-7.3 m and median OS 6.2 m, 95% CI 4.8-7.3 m) and without a KRAS mutation (median PFS 6.1, 95% CI 5.0-7.2m and median OS 8.9 m, 95% CI 6.4-11.4).

Conclusion: In our series KRAS mutation was not predictive for response to platinum containing CT in pts with advanced NSCLC. Furthermore, KRAS mutation was not prognostic. Additional data on specific KRAS mutations will be presented.

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 340. doi:10.1158/1538-7445.AM2011-340
Abstract 3414: EMT-associated genes are altered in giant cell tumor of bone
Authors: Rahal Paula , Babeto Erica , de Freitas Calmon Marilia , Conceição Andre , Zuccari Débora
Published: June 2012

The giant cell tumor of bone (GCT) is a benign bone-destructive neoplasm, with uncertain biological behavior, with features such as a large number of multinucleated giant cells and aggressive behavior. The local recurrence of GCT is often observed, especially in the first three years after treatment, giving a rate of recurrence ranging from 20% to 50% of cases. Further aggravating the recurrence, is the fact that after a relapse, patients often also presents metastasis in other organs, mainly in the lungs. In this study we investigate the expressions of epithelial and mesenchymal (EMT) markers (ADAM23, CDH1, CDH3, MMP14, p16, TIMP2 and VIM) in GCTs by quantitative qPCR. The expression of the MMP14, TIMP2 and VIM genes increased in more than 96% samples and ADAM23, CDH1, CDH3 and p16 genes had expression reduced in more than 91% of GCTs samples. Since ADAM23, CDH1, CDH3 and p16 have CpG islands in their promoter regions and low expression in tumor tissue, their methylation patterns were analyzed by MSP-PCR. For ADAM23 and p16 genes were detected a high frequency of hypermethylation. The lower expression of ADAM23 and p16 are directly related to the recurrence and metastasis in GCTs. The genes MMP14, TIMP2 and VIM are related to angiogenesis, degradation of extracellular matrix and cell migration and presented with increased expression in GCT, also confirmed by immunohistochemistry. These observations suggest that dysregulation of these genes may be a key factor responsible for the initiation and perpetuation of EMT in GCT, leading to cellular migration and malignant transformation, contributing to development of metastases and tumor progression.

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 3414. doi:10.1158/1538-7445.AM2011-3414
Abstract 3415: Diverse roles for Snail transcription factor in epithelial-mesenchymal transition (EMT) and neuroendocrine differentiation (NED) in human prostate cancer
Authors: McKeithen Danielle , Graham Tisheeka , Morrissey Colm , Zhang Xiaotun , Chung Leland W K , Odero-Marah Valerie
Published: June 2012

Snail transcription factor induces epithelial-mesenchymal transition (EMT) via decreased cell adhesion-associated molecules like E-cadherin, and increased mesenchymal markers like vimentin. We previously established Snail-mediated EMT model utilizing androgen-dependent LNCaP cells. These cells express increased vimentin protein and relocalization of E-cadherin from the cell membrane to the cytosol. Interestingly, Snail transfection in LNCaP cells resulted in cells acquiring a neuroendocrine-like morphology with long neurite-like processes. We tested for expression of neuroendocrine markers neuron specific enolase (NSE) and chromogranin A (CgA) by Western blot analysis, and performed proliferation assays to test for paracrine cell proliferation. LNCaP cells transfected with Snail displayed increase in the neuroendocrine markers, NSE and CgA as well as translocation of androgen receptor to the nucleus. We found that LNCaP C-33 cells that have been previously published as a Neuroendocrine Differentiation (NED) model exhibited increased expression levels of Snail protein as compared to LNCaP parental cells. Functionally, conditioned medium from the LNCaP-Snail transfected cells increased proliferation of parental LNCaP and PC-3 cells. In addition, NED in LNCaP-C33 cells or that induced in parental LNCaP cells by serum starvation could be inhibited by knockdown of Snail with siRNA. Knockdown of NSE and CgA with siRNA abrogated Snail induced paracrine cell proliferation in parental LNCaP and PC3 cells. Immunohistochemical staining of prostate cancer patient tissue revealed increased expression of Snail with prostate cancer progression and bone metastasis. Higher Snail staining was observed in primary prostate cancer tissue containing a neuroendocrine component (identified by CgA staining) as compared to primary cancer with no neuroendocrine differentiation. However, in metastatic tissue, Snail was highly expressed in both neuroendocrine and non-neuroendocrine cancer tissue. Overall our data provide evidence that Snail transcription factor may promote tumor aggressiveness in the prostate cancer cells through multiple processes; induction of EMT may be required to promote migration, while NED may promote tumor proliferation by a paracrine mechanism. Therefore, therapeutic targeting of Snail may prove beneficial in not only abrogating EMT but also NED.

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 3415. doi:10.1158/1538-7445.AM2011-3415
Abstract 3425: Interleukin-27 inhibits epithelial mesenchymal transition in lung carcinogenesis
Authors: Lee Jay M. , Zhang Ling , Kachroo Puja , Cui Xiaoyan , Hazra Saswati , Walser Tonya C. , Minna John D. , Sharma Sherven , Dubinett Steven M.
Published: June 2012

Interleukin-27 (IL-27), the newest member of the IL-6/IL-12 family, is secreted by antigen presenting cells and has anti-tumor activity through its activation of the JAK-STAT pathway. Transcriptional factors STAT1 and STAT3 have been implicated in the balance of tumor suppressor and oncogenic functions. Epithelial mesenchymal transition (EMT) is a process whereby cells undergo changes from a highly polarized epithelial phenotype to a mesenchymal, migratory phenotype, which plays an important role in tumor metastasis. The role of IL-27 in EMT was evaluated in lung carcinogenesis. Human bronchial epithelial cells (HBECs), genetically modified HBEC to over-express Snail and K-ras (HBEC-Snail, HBEC-K-ras), and non-small cell lung cancer (NSCLC) lines (A549, H2122) were treated with IL-27 at multiple time points. Phosphorylation of STAT1 and STAT3 measured by Western Blot demonstrated activation of both pathways in these cell lines treated with IL-27. However, the STAT1 pathway was attenuated in HBEC-K-ras cells exposed to IL-27. Western analysis of markers for epithelial to mesenchymal transition (EMT) (E-cadherin, γ-catenin, N-cadherin, Vimentin, and Snail) showed up-regulation of E-cadherin and γ-catenin, important for the epithelial phenotype, which peaked 8 hours after IL-27 exposure, while markers important for the mesenchymal phenotype (N-cadherin, Vimentin, and Snail) were all down-regulated at similar time points. The addition of a STAT3 inhibitor to IL-27 treatment led to continued down-regulation of Vimentin beyond 8 hours. A wound healing functional assay to study cell migration after IL-27 treatment of cancer cell lines for up to 48 hours, demonstrated overall decrease in cell migration. In summary, IL-27 inhibits epithelial mesenchymal transition in NSCLCs and oncogenically manipulated HBECs through the regulation of STAT1 and STAT3 pathways.

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 3425. doi:10.1158/1538-7445.AM2011-3425
Abstract 3429: Mesenchymal hepatocellular carcinoma cells acquire cancer stem cell characteristics through Snail1
Authors: Dang Hien , Ding Wei , You Hanning , Emerson Dow , Rountree Carl B.
Published: June 2012

Hepatocellular carcinoma (HCC) is the third leading cause of cancer mortality and the incidence of HCC is rising in the United States. Cancer stem cells (CSCs) are a subset of neoplastic cells that possess distinct survival mechanisms and plasticity crucial for tumor maintenance and propagation. The induction of epithelial-mesenchymal-transition (EMT) by Transforming Growth Factor-β (TGF-β) has been linked to the acquisition of CSC characteristics in breast cancer. In patients with HCC, either a CSC or EMT phenotype correlates with a worse prognosis. In this report, we aim to link EMT to CSCs and to further elucidate the mechanism by which cells acquire CSC characteristics during EMT. We utilized human (Huh7, MHCC97-L) and murine (P2E, P2M; Ding et al Hepatology. 2010) liver cancer cell lines, and analysis included gene and protein expression, FACS, migration and invasion assays, and chemotherapy resistance assay. Human mesenchymal MHCC97 cells demonstrate decreased E-cadherin, increased Snail1, and enhanced migration and invasion compared to epithelial Huh7 cells. MHCC97 cells demonstrate significant upregulation of stem cell markers (Oct4, Nanog, BMI-1, and CD44; p<0.01), increased CD44high population (95% vs. 2% Huh7, p<0.01), resistance to chemotherapy, resistance to TRAIL-induced apoptosis, increased tumor-sphere formation and slower cell cycle compared to epithelial Huh7 cells. We recently reported EMT in murine epithelial liver cell lines, P2E and P2M (post EMT). Mesenchymal P2M cells demonstrate loss of E-cadherin, gain of Snail1, increased tumor-sphere formation, and increased expression of stem cell markers (Nanog, Oct4, CD44, and EpCam; p<0.001). As TGF-β induces EMT through Snail1, we hypothesize that Snail1 signaling plays a role in the induction of an EMT and CSC phenotype in HCC. TGF-β treatment, epithelial P2E cells demonstrate fibroblastic-like morphology, 14-fold increase in Snail1 (p<0.01) and 8 fold increase in Nanog expression (p<0.01), increased tumor-sphere formation and migration. siRNA knockdown of Snail1 in mesenchymal P2M cells resulted in significant decrease in EMT and CSC characteristics, with decreased tumor-sphere formation, decreased migration, and decreased stem cell gene expression (Nanog and CD44, p<0.01). In conclusion, both murine and human mesenchymal HCC cells possess multiple CSC characteristics compared to epithelial counterparts. The transient knockdown of Snail1 in mesenchymal HCC cells results in the reversal of EMT as well as loss of CSC characteristics. We propose that Snail1 may play an important role in the regulation of EMT and CSC phenotypes.

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 3429. doi:10.1158/1538-7445.AM2011-3429
Abstract 343: Molecular markers for detection of micrometastases in regional lymph nodes from patients undergoing surgery for non-small cell lung cancer
Authors: Nordgård Oddmund , Ulas Mona , Singh Gurpartap , Fleischer Thomas , Sagerup Camilla M. , Småland Rune , Brustugun Odd Terje , Helland Åslaug
Published: June 2012

PURPOSE: Lung cancer retains its status as the most common cancer in the world, with an estimated 1.5 million cases diagnosed in 2007, accounting for about 12% of all cancers. Identifying microscopic lynph-node metastases, could have clinical importance in selecting patients for adjuvant treatment. The purpose of this study wasis to Iidentify and evaluate molecular markers for detection of micrometastases in regional lymph nodes from patients undergoing surgery for non-small cell lung cancer.

PATIENTS AND METHODS: Candidate micrometasis markers were selected based on digital transcript profiling in EST and SAGE databases, searching for transcripts with high levels in lung cancers and very low levels in normal lymph nodes. Surfactant protein C (SFTPC) and cytokeratin 19 (CK19) mRNA levels in tumor biopsies and resected regional lymp nodes (N=38) from 24 patients undergoing surgery for non-small cell lung cancer were measured by quantitative RT-PCR. A control material consisting of 22 normal mesenterial lymph nodes from patients undergoing surgery for benign colon disorders was analyzed in parallell.

RESULTS: The median relative CK19SFTPC mRNA level in the non-small cell lung tumor biopsies were 446 times higher than the highest level in normal control lymph nodes. SFTPC mRNA was not detectable in the normal lymph nodes, whereas the tumor levels were high (Cq-values ranging from 17-25). Using the highest CK19 mRNA level in the normal lymph nodes as a cut-off, four out of five (80%) lymph nodes from patients with known lymph node metastases (by routine histology) had elevated CK19 levels. Accordingly, 5 (15%) of 33 lymph nodes from patients without known metastases (node-negative) had elevated levels of CK19 mRNA. SFTPC mRNA were detectable in 31 of the 33 lymph nodes from node-negative patients, although the median relative level was 7.1 times higher in the 5 lymph nodes from node-positive patients (P=0.002, Mann-Witney test). Elevated CK19 and SFTPC mRNA levels in regional lymph nodes without known metastases may reflect the presence of micrometastases.

CONCLUSION: SFTPC and CK19 mRNA are promising surrogate markers for detection of micrometastases in regional lymph nodes from non-small cell lung cancer patients. However, clinical follow-up in a larger cohort is needed to eludicate the prognostic value of such findings.

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 343. doi:10.1158/1538-7445.AM2011-343