bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3319: CD44 standard isoform regulates the mesenchymal phenotype in hepatocellular carcinoma cells, and associates with a poor prognosis in patients.
Authors: Mima Kosuke , Okabe Hirohisa , Ishimoto Takatsugu , Beppu Toru , Baba Hideo
Published: August 2012

Purpose: Cancer stem cells (CSCs) in many tumors have been identified using CD44, either individually or in combination with other markers and are involved in tumor progression and metastasis. Epithelial-mesenchymal transition (EMT) is known to induce cancer invasion, metastasis, and CSCs properties. However, the underlying molecular mechanisms which integrate EMT and CSCs properties remain largely unknown. In this study, we test the hypothesis that CSCs markers play an important role in regulating EMT in hepatocellular carcinoma (HCC). Experimental Design: The relationship between the CSCs markers and EMT markers was investigated in HCC cell lines. The EMT markers, invasive phenotype, the relevant transcriptional factors and signaling pathways induced by CD44 were examined in vitro. The clinicopathological importance of CD44 standard isoform (CD44s) expression was analyzed by an immunohistochemical analysis, and the correlation of CD44s and EMT markers was determined in 150 patients with hepatocellular carcinoma. Results: HCC cell lines with high CD44 expression showed decreased E-cadherin expression and increased vimentin expression. CD44s mRNA was dominant form of CD44 mRNA present in HCC cell lines with the mesenchymal phenotype. Overexpression of CD44s in HCC cells induced the mesenchymal phenotype and increased tumor cell invasion, whereas knockdown of CD44s in HCC cells led to loss the mesenchymal phenotype and decreased tumor cell invasion. CD44s expression was associated with E-cadherin low expression (P=0.039) and vimentin high expression (P<0.001). CD44s expression was related to a large tumor size (P=0.003), multiple tumors (P=0.032), and poor tumor differentiation (P=0.020), and a shorter disease-free (P=0.023) and overall survival (P=0.013) in patients with HCC. Conclusions: our study will be valuable for understanding the relationship between CSCs and EMT, and the identification of new treatment targets for future HCC treatment.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3319. doi:1538-7445.AM2012-3319
Abstract 333: SFRP4 modulates EMT, cell migration and downstream Wnt signalling in epithelial ovarian cancer.
Authors: Ford Caroline E. , Jary Eve , Ma Sean , Nixdorf Sheri , Heinzelmann-Schwarz Viola , Ward Robyn L.
Published: August 2012

Epithelial ovarian cancer is the fifth most common cause of death from all female cancers. This poor overall prognosis results in part from late diagnosis and a current lack of targeted therapy, stemming from the current limited understanding of the initiating events of ovarian carcinogenesis. There is a critical need for the identification of ovarian cancer pathways and targets to aid in diagnosis, as prognostic indicators and as targets for novel therapies. Wnt signalling is essential for crucial components of carcinogenesis and metastasis including differentiation, polarity, migration, adhesion and survival, and hence the role of Wnt signalling in human cancer is increasingly being investigated along with strategies to target pathway components. The Wnt pathway is regulated at multiple levels, with the Wnt antagonists or “gatekeeper proteins” receiving attention in recent years, due to their frequent loss in cancer. Secreted frizzled receptor proteins (SFRPs) are extracellular inhibitors of Wnt signalling that act by binding directly to Wnt ligands or Frizzled receptors, and have been shown to function as tumour suppressors in other cancer types. We have previously shown that one of these inhibitors, SFRP4, is progressively lost in more aggressive ovarian cancer phenotypes, such as Type II cancers (Jacob et al, 2011). Furthermore, patients lacking SFRP4 expression had a poorer prognosis than those expressing SFRP4. Here, we investigated the functional consequences of SFRP4 modulation in ovarian cancer cell lines. Re-expression of SFRP4 resulted in decreased expression of downstream components of Wnt signalling, inhibition of cell migration, and inhibition of epithelial to mesenchymal transition (EMT). In parallel, knockdown of SFRP4 resulted in ovarian cells upregulating markers of EMT. These results indicate that modulation of an upstream gatekeeper of Wnt signalling can have dramatic downstream effects on ovarian cancer cell behaviour, as mediated through EMT. This raises the possibility that SFRP4 may be a suitable marker of ovarian carcinogenesis, and that targeting the Wnt signalling pathway may have potential as an ovarian cancer treatment.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 333. doi:1538-7445.AM2012-333
Abstract 3348: Pax8-PPARδ fusion protein activates β-catenin/TCF pathways
Authors: Vu-Phan Dang L. , Yu Jingcheng , Koenig Ronald J. , Wicha Max S.
Published: August 2012

Pax8-PPARδ Fusion Protein (PPFP) occurs in ∼35% of follicular thyroid carcinomas. Expression of PPFP in the non-transformed rat thyroid cell line PCCL3 conferred on the cells the ability to invade through matrigel and to form colonies in anchorage independent conditions. PPFP also increased the percentage of cells that have activated β-catenin/TCF. We transfected control and PPFP expressing PCCL3 thyroid cell lines with two different β-catenin/TCF-GFP reporter systems. We then sorted a population of GFP positive cells and expanded them to obtain 100% transfected cell lines. These GFP positive cells gave rise to a new heterogenous population with a stable equilibrium between GFP positive, β-catenin/TCF active cells and GFP negative, β-catenin/TCF inactive cells. PPFP expressing cell lines contained more than twice the percentage of GFP positive cells compared to control cell lines. GFP positive PPFP expressing thyroid cells were enriched for cells exhibiting the transformed phenotypes previously observed in the unsorted, untransfected cell lines. Sorted β-catenin/TCF active single cells gave rise to heterogenous clonal populations containing both β-catenin/TCF active and negative cells. Clonal populations derived from β-catenin/TCF inactive cells remained purely negative. Clonal populations derived from GFP positive cells were twice as enriched for anchorage independent colony forming cells than GFP negative clones. More strikingly, GFP positive clones were 5-10 times more invasive than negative clones, which exhibited the same invasive potential as control cells. Treatment with PPARδ full or partial agonists further increased the β-catenin/TCF population in PPFP expressing cells. Again, the effects were confined to populations containing β-catenin/TCF active cells. Co-treatment with PPARδ antagonists reversed the effects of PPARδ agonists. These data suggest that β-catenin/TCF active cells have properties of cancer stem cells and that PPFP acts on this population to expand the stem cell like fraction.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3348. doi:1538-7445.AM2012-3348
Abstract 3350: Implications of the STAT6 pathway by interleukin-4 in prostate cancer
Authors: Erb Holger H. , Santer Frédéric R. , Culig Zoran
Published: August 2012

Cancer-related inflammation (CRI) can promote or inhibit tumor development, growth and progression. A key role in CRI may be the infiltration of immune cells. During the inflammation process immune cells are recruited to the tumor. They produce cytokines and inflammatory molecules like reactive oxygen and nitrogen species. However, in prostate Cancer (PCa) the role of inflammation is not well understood. Meta-analysis conducted on case-control studies showed an association of prostatitis and the risk of prostate cancer. Moreover, clinical data showed that several cytokines e.g. interleukin (IL)-6, IL-8 and IL-4 are significantly elevated in sera of patients with CRPC. In this study we investigated the role of short-term and long-term IL-4 treatment on the castration resistant prostate cancer lines PC3, Du145 and LNCaP-IL6+. For short-term treatment conditions cells were incubated with increasing concentrations of IL-4 (0.1-10 ng/ml) for 96 h in full growth medium. Neither proliferation rate nor cell-viability of the three cell lines were changed under these conditions. On the other hand, from the known IL-4 downstream targets only phosphorylation of STAT6 was readily detectable after IL-4 treatment, whereas phosphorylation status of the MAP kinases ERK1/2 and AKT was not affected. Among the STAT6 regulated genes only SOCS-1 was upregulated after IL-4 treatment. For long-term treatment conditions PC3, Du145 and LNCaP-IL-6+ cell lines were treated with 5 ng/ml IL-4 over a period of 30 passages. Interestingly, a morphologic change of the PC3 cell line to a more dedifferentiated phenotype was observable after 10 passages of IL-4 treatment and peaked after 20 passages with 30-50% of all cells showing this morphology. In order to evaluate possible effects of IL-4 on stem-cell like phenotype, we analyzed the IL-4 treated PC3 cell line for the presence of stem cell markers CD44, CD133 and integrin α2 by FACS. Indeed, high expression of CD44 and integrin α2 was observable in a subpopulation of PC3 cells. Expression of CD133 was negative due to hypermethylation of the gene. The percentage of marker-positive cells was increasing with passage, showing that IL-4 long-term treatment might be favorable for cells with cancer stem cell-like properties. Collectively, our results show that IL-4 long-term treatment might promote cells with stem cell-like characteristics and therefore influence the progression of the cancerous disease. This work was funded by the European Union FP7 Marie Curie Initial Training Network PRO-NEST.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3350. doi:1538-7445.AM2012-3350
Abstract 3353: Nodal signaling mediates the aggressive, tumorigenic phenotype in breast cancer cells
Authors: Kirsammer Gina , Strizzi Luigi , Gilgur Alina , Margaryan Naira , Kirschmann Dawn , Seftor Elisabeth , Hendrix Mary JC
Published: August 2012

The embryonic morphogen Nodal is a potent inducer of EMT and critical mediator of stem cell pluripotency in the developing embryo, but is not expressed in normal adult tissues. Intriguingly, our lab has recently shown that Nodal, a TGF-beta family member, is in fact secreted by melanoma and breast cancer cells and that Nodal expression positively correlates with tumor grade in human patient samples. Our current studies demonstrate a positive role for Nodal in regulating breast cancer cell renewal and aggressiveness. Specifically, short hairpin (shRNA) knockdown of Nodal expression in human breast cancer cell lines diminishes proliferation, invasion and clonogenicity of these cell lines in vitro and dramatically curtails tumor formation in a mouse xenograft model. Additionally, our data suggest that these effects may be caused, in part, by alterations in the expression of adhesion receptors known to function in tumor engraftment and metastasis. Taken together, our data suggest that Nodal signaling may facilitate the acquisition of self-renewal and migratory capabilities in breast cancer cells and represent a novel therapeutic target for disrupting these processes in breast cancer cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3353. doi:1538-7445.AM2012-3353
Abstract 3358: 1α,25-dihydroxyvitamin D3 inhibits the proliferation of cultured glioblastoma multiforme cells
Authors: Pinskey Justine M. , Winn Robert J.
Published: August 2012

Glioblastoma multiforme (GBM) is a common, aggressive type of brain tumor with a median survival of only fifteen months. Brain tumor stem cells (BTSCs) within GBM tumors resist standard treatments, initiate recurrence, and pose a significant challenge for GBM treatment. This study examined Gli1 expression and proliferation of GBM cells in vitro to evaluate the effects of 1α,25-dihydroxyvitamin D3 (vitamin D3). Vitamin D3 is a safe, natural inhibitor of the hedgehog signaling pathway–a mechanism essential to BTSC function. Immunocytochemistry demonstrated that both established GBM cell lines and GBM-derived BTSCs expressed Gli1, indicating hedgehog signaling pathway activity within the cell populations. Vitamin D3 treatment significantly reduced cell proliferation, both in GBM cell lines and GBM-derived BTSCs. Because of active vitamin D3's environmental instability, an in vivo model might provide a better indication of its anti-tumor effects. Ultimately, vitamin D3 may enhance standard GBM treatments by inhibiting hedgehog-expressing cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3358. doi:1538-7445.AM2012-3358
Abstract 336: 4-methylumbeliferone, an HA synthesis inhibitor, inhibits EMT determinants in bladder cancer cell lines
Authors: Yates Travis , Garcia-Roig Mike , Veerapen Muthu K. , Benitez Anaid , Lokeshwar Vinata
Published: August 2012

INTRODUCTION AND OBJECTIVE: Elevated Hyaluronic Acid (HA) levels are a molecular determinant of bladder cancer (BCa) metastasis. Furthermore, HA, HYAL-1 hyaluronidase and HA-synthesizing enzymes are potential markers for BCa diagnosis and predicting prognosis. 4-Methylumbelliferone (4-MU) is an orally bioavailable dietary supplement that inhibits HA synthesis. Epithelial mesenchymal transition (EMT) is the hallmark of invasion and metastasis, in which cancer cells acquire an invasive phenotype. Since HA promotes tumor metastasis, we evaluated the antitumor effects of 4-MU in vitro and in vivo and examined whether 4-MU can reverse the EMT by inhibiting HA synthesis. METHODS: Quantitative PCR was used to measure mRNA expression of EMT genes (β-catenin, Twist, and Snail) in 66 bladder tissue specimens (27 normal; 39 tumor); follow-up: 26±4.3 months; median 20 months. The effect of 4-MU (0.2 - 0.6 mM) on cell proliferation, apoptosis, intracellular signaling, and the expression of HA receptors and EMT genes were examined in BCa cell lines, 253J-Lung and HT1376. Effect of 4-MU on tumor growth was analyzed in subcutaneous xenografts. RESULTS: Among the EMT genes, both Snail and Twist were differentially expressed in BCa tissues when compared to normal bladder (P<0.001). Furthermore, Twist expression significantly correlated with metastasis (+: 0.25+0.15, -: 0.12+0.14; P=0.028). β-catenin expression negatively correlated with survival (chi square=4.3, p=0.038). 4-MU inhibited proliferation and induced apoptosis in the BCa cells. Following a 48 h exposure at the IC50 for HA synthesis (0.4 mM), 4-MU inhibited proliferation by 61% and increased apoptosis by 73%. 4-MU induced caspase-8, -9 and -3 activation and up-regulation of Fas and FADD. 4-MU downregulated the expression of HA-receptors - CD44, RHAMM, EMT promoters - β-catenin, Snail and Twist by 4-10-fold, but increased the expression of E-cadherin by 2-fold, suggesting EMT-reversal. In xenograft studies, 4-MU significantly decreased tumor growth (> 3-fold) when treatment was started either on the day of tumor cell injection or after tumors became palpable. No weight loss or serum or organ toxicity was observed in treated mice. Tumors showed reduced microvessel density (∼3-fold) and HA expression but increased TURNEL positive cells. CONCLUSION: This is the first study that shows inhibition of HA synthesis by an orally bioavailable, non-toxic supplement potentially reverses EMT and has potent anti-tumor activity. Support: R01 CA 123063-04 (VBL); R01 CA 72821-11 (VBL).

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 336. doi:1538-7445.AM2012-336
Abstract 3381: Photochemical internalization (PCI) of immunotoxins targeting cancer stem cell markers
Authors: Bostad Monica , Berg Kristian , Høgset Anders , Selbo Pål Kristian
Published: August 2012

The cancer stem cell (CSC) theory indicates that only a subset of the cancer cells is capable of tumor-initiation, self renewal and differentiation. CSC is also thought to be treatment resistant and responsible for recurrence. Different antigens are being used to identify CSC markers and are potential targets for CSC-based therapy. Due to the expression of the same antigens on normal stem cells it is of paramount importance that CSC-targeting cytotoxins are specifically and efficiently delivered to malignant tissues. Photochemical internalization (PCI) is a method for specific delivery of membrane impermeable macromolecules from endocytic vesicles to the cytosol of targeted cells. In this study we show that PCI increase the cytotoxic effect of immunotoxins (IT) targeting stem cell markers on different cell lines. The IT consists of a CSC-targeting mAb biotinylated to the ribosome inactivating plant (RIP) toxin saporin. TPCS2a-PCI of one of the ITs showed to be very cytotoxic at sub picomolar levels. Cytotoxicity of the IT was blocked by using excess concentration of unconjugated Ab. In addition, no difference in cytotoxicity was obtained between mAb-toxin and toxin alone in marker negative cell lines, confirming specificity. In order to asses enrichment of CSC-activity in vitro we used flow cytometry to sort cells based on the degree of expression of CSC-markers and compared the ability of different cell fractions to establish colonies in both 2D and 3D cell cultures growing in serum-free stem cell media. Cells with high expression of CSC-markers had significantly higher capacity to initiate colonies than low expressing cells. CSC enrichment was confirmed by an in vivo limiting dilution assay. The present study demonstrates that PCI is an efficient method for selective killing of cancer cells expressing stem cell markers and will be further explored for the targeting of tumors that express these markers.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3381. doi:1538-7445.AM2012-3381
Abstract 3849: Triptolide reduces activity of RNA polymerase II
Authors: Yang Shanmin , Huang Luqiang , Chen Chun , Zhang Mei , Zhang Zhenhuan , Han Deping , Hong Jinsheng , Yin Liangjie , Zhang Bingrong , Zhang Amy , Vidyasagar Sadasivan , Okunieff Paul , Zhang Lurong
Published: August 2012

Triptolide (TPL) possesses potent anticancer and anti-inflammatory effects. Our previous study demonstrated that radiation-induced pulmonary fibrosis in mice can be significantly reduced by TPL treatment (0.25 mg/kg/every 3 days for 1-3 months via intravenous injection). Treatment was associated with the reduction of a panel of inflammation molecules, as evidenced by a membrane array of 62 inflammatory molecules. We found that TPL has a high level of biointeraction with RNA polymerase II (Pol II). Pol II is a major enzyme complex responsible for the production of mRNA and could explain improved tumor response and reduced normal tissue toxicity. The mRNA of interleukin-1 beta (IL-1α), tumor necrosis factor-alpha (TNFα), IL-6, and Cox-2 were used as downstream products of Pol II. Alpha-amanitin (a specific inhibitor of Pol II) and actinomycin D (a pan Pol inhibitor) were used as positive controls. The results showed that: 1) Pol II was suppressed as early as 1 hour after TPL was added to cells; 2) TPL (effective at ng/ml levels) is more potent than α-amanitin and actinomycin D (effective at µg/ml levels) at reducing housekeeping genes L32 and S14; 3) the suppression of Pol II by TPL was dose dependent; and 4) TPL inhibited radiation-induced mRNA of IL-1α and TNFα at 3 hours after irradiation and IL-6 and Cox-2 at 8 hours after irradiation. It is unknown whether these early effects of TPL on Pol II impact either tumor response or normal tissue complications.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3849. doi:1538-7445.AM2012-3849
Abstract 3851: Killing of Kras mutant colon cancer cells by the αGBP cytokine, a physiological PI3K inhibitor therapeutically effective in vivo
Authors: Mallucci Livio , Lotti Lavinia V. , Mariani-Costantini Renato , Wells Valerie , Zicha Daniel
Published: August 2012

Activating Kras mutations are the most frequent oncogenic mutations in human cancer. Numerous downstream pathways responsible for cell proliferation and cell survival have been shown to be deregulated by oncogenic Kras but there are no effective targeted therapies to date for Kras driven oncogenesis. Mutant-activated Kras defines a subset of patients for whom prognosis is poor and therapeutic options are limited. We demonstrate that αGBP, a cytokine produced by CD4+ and CD8+ activated T cells, which operates by inhibiting class IA and class IB PI3K, is a potent activator of apoptosis in Kras-mutant colorectal cancer cells, even when co-harboring mutant-activated PIK3CA. Mechanics initiate with inhibition of PI3K activity and Rac-independent actin reorganisation assignable to phosphoinositide changes at the plasma membrane. Cyclin E deregulation, arrest of DNA synthesis and Chk2 activation underscore events critical to the activation of intrinsic apoptosis. Clustering of CD95/Fas death receptors underscores events critical to the activation of extrinsic apoptosis. In nude mice we present the first evidence that tumor development is strongly inhibited. This first demonstration of therapeutic efficacy against Kras-mutant colon cancer suggests that Hu-r-αGBP may be therapeutically effective against other cancers harbouring activating Ras mutations. Wells, V. and Mallucci, L. (2009). Phosphoinositide 3-kinase targeting by the β-galactoside binding protein cytokine negates akt gene expression and leads aggressive breast cancer cells to apoptotic death. Breast Cancer Research 11, R2, 1-10. Wells, V., Downward, D. and Mallucci, L. (2007). Functional inhibition of PI3K by the αGBP molecule suppresses Ras-MAPK signalling to block cell proliferation. Oncogene 26, 7709-7714. Mallucci, L. and Wells, V. (2007). Alternative use of signaling by the αGBP cytokine in cell growth modulation and cancer control. From surveillance to therapy. In: Apoptosis, Cell Signaling and Human Diseases. Ed. R. Srivastava. The Humana Press Inc. Vol. I, 203-216. Patents US patent no. 7,994,113 granted August 9th 2011 PCT WO 2008/152392: Europe, Canada, Australia, Japan, published

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3851. doi:1538-7445.AM2012-3851
Abstract 3855: Inhibitors of apoptosis (IAPs) in gastrointestinal stromal tumors
Authors: Falkenhorst Johanna , Mühlenberg Thomas , Simon Susanne , Ecken Sonja , Henze Joern , Grabellus Florian , Breitenbücher Frank , Schuler Martin , Fletcher Jonathan A. , Bauer Sebastian
Published: August 2012

Introduction Gastrointestinal stromal tumors (GIST) are characterized by gain-of-function-mutations of c-KIT or PDGFRA. Despite long-lasting remissions with the KIT inhibitor imatinib (IM) most patients eventually progress. While inhibitors of apoptosis proteins (IAPs) may confer resistance in other cancers little is known about the role of IAPs in GIST. Material and Methods IAP expression (cIAPs, XIAP, survivin) in patient samples (TMA, n=163), IM-sensitive (GIST-T1, GIST882, GIST430) and IM-resistant (GIST48B, GIST48) GIST cell lines (RT-PCR, western blot) was analyzed. The impact of KIT and growth inhibitory drugs on IAP expression (24h of treatment) was determined by western blot. Biological consequences of IAP modulation (YM155: transcriptional repressor of survivin; Embelin: biochemical XIAP inhibitor; lentiviral shRNA knockdown [KD] of survivin, XIAP) were investigated alone and in combination with KIT-related pathway inhibitors (IM, LY294002, 17-AAG). Results cIAPs, XIAP and survivin were expressed at various levels in all KIT-positive and negative GIST cell lines. Resistant cell lines did not exhibit higher levels of IAPs. Survivin expression was found to vary over time in GIST-T1 and 48B (2- and 4-fold within 5 days. Survivin expression (immunoreactivity score α3) was found in 40% of GIST patients. PI3K inhibition led to survivin downregulation of 20 to 65% (24h) in all tested cell lines whereas KIT inhibition (IM, 24h) reduced survivin and XIAP expression only in GIST-T1 (52%). The protein levels of both XIAP and survivin were decreased following HDAC (SAHA: 44-71%, except GIST430) and MDM2 inhibition (survivin: 40 - 90%). YM155 downregulated survivin and exhibited proapoptotic and antiproliferative effects in all cell lines at low nM levels (IC50: GIST-T1: 4nM, GIST48P: 40nM, GIST48B, GIST 882: 80nM) except GIST430 (IC50>1µM). No antagonistic effects were seen with combinational treatment. Embelin treatment resulted in IC50 levels of 40µM - 300µM while lacking a specific XIAP inhibition. A stable XIAP KD was established in GIST-T1 resulting in slower growth (25% in 3d). Conclusion IAPs are commonly expressed in GIST cell lines regardless of resistance to IM. Expression levels are mostly independent from KIT inhibition but proapoptotic effects from HDAC and MDM2 inhibitors may in part be conferred by IAPs. The survivin repressor YM155 showed antiproliferative and proapoptotic effects at levels that are therapeutically achievable suggesting a potential therapeutic role in GIST.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3855. doi:1538-7445.AM2012-3855
Abstract 3858: Gains in FGF19 are predictive of response to the fibroblast growth factor receptor (FGFR) small molecule tyrosine kinase inhibitor BGJ 398 in vitro
Authors: Finn Richard S. , Aleshin Alex , Zhao Danyun , Anderson Lee , Ginther Charles , Dering Judy , Shi Michael , Porta Diana Graus , Busuttil Roanld W. , Slamon Dennis J.
Published: August 2012

Background: Hepatocellular carcinoma (HCC) is a leading cause of cancer death world-wide. Sorafenib, a multi-targeted small molecule inhibitor of the vascular endothelial growth factor (VEGF) receptor and other kinases is the only approved systemic agent in advanced HCC. Despite multiple studies defining the molecular heterogeneity of HCC, clinical trials of new agents do not pre-select for predictive markers. The fibroblast growth factor (FGF) receptor consists of 4 receptor tyrosine kinases and over 20 ligands. Differential expression and activation of these receptors and ligands have been implicated in oncogenesis and the pathogenesis of HCC (Sawey 2011). BGJ398 is a selective small molecule tyrosine kinase inhibitor of the FGFR family of receptors 1-4. Given the molecular diversity of HCC, we hypothesized that there would be a subset of HCC that would be more dependent on FGFR inhibition and used cell line models to identify predictive markers of response to BGK 398. Methods: 20 human HCC cell lines were used. In vitro dose response curves were generated using direct cell count assays over 6 days. IC 50 values were calculated from the dose response curves. Genomic data on the cell lines was derived using the Agilent 105K CGH platform. These data were analyzed in the context of the sensitivity data to BGJ 398. Western blot analysis was performed for effects of BGJ398 on phospho-FRS2Δ (tyr-196), a downstream signaling molecule for FGFR activation. Flow cytometry was performed for effects of BGJ 398 on the cell cycle and apoptosis. Results: There was differential sensitivity to BGJ 398 across the HCC cell lines. 3 cell lines were identified as having IC 50 values less than 1 μM versus the remaining cell lines which were all >1 uM. When analyzed in the context of the CGH data, all 3 cell lines had gains in FGF 19 of log2 ratio of 0.9 to 2.6. BGJ inhibited FRS2α activation in all cell lines tested. Flow cytometry revealed the BGJ induced cell cycle arrest in sensitive lines without significant apoptosis. Conclusions: Gains in FGF19 are predictive for growth inhibitory effects of BGJ398 in human HCC cell lines in vitro and provide rationale for patient selection in clinical trials.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3858. doi:1538-7445.AM2012-3858
Abstract 3889: Substituted pyrrolo[2,3-d]pyrimidines as water soluble microtubule targeting agents
Authors: Gangjee Aleem , Lin Lu , Mooberry Susan L. , Hamel Ernest
Published: August 2012

Microtubules play critical roles in cell functions, and microtubule targeting agents have been successfully developed as anti-cancer drugs. Based on the binding sites, microtubule targeting agents are divided into three broad classes: vinca alkaloid, taxane and colchicine site binders. Vinca alkaloids and taxanes, represented by vinorelbine and paclitaxel, respectively, have been widely used in cancer chemotherapy. However, drug resistance associated with over-expression of Pgp or αIII-tubulin has greatly reduced the therapeutic value of these drugs (e.g., paclitaxel is over 800-fold less active against Pgp over-expressing cancer cells). Another persistent issue with vinca alkaloids and taxanes is their poor water solubility. Administration of paclitaxel needs to be performed by trained personnel with special reagents and equipment. Colchicine site binders, such as colchicine or combretastatin A-4 (CA4), have not received clinical approval for wide use in the treatment of cancer. Novel water soluble colchicine site binding agents are potentially valuable alternatives for use alone or in combination with existing microtubule targeting drugs, especially in overcoming drug resistance. We previously reported a series of substituted pyrrolo[2,3-d]pyrimidine compounds as water soluble, colchicine site binding agents. One of the highly active compounds has shown sub-micromolar to nanomolar inhibitory activities against the NCI-60 panel cell lines. The same compound exhibits enhanced or equal inhibition against Pgp and αIII-tubulin over-expressing cell lines. In a continuing effort to find new agents with these properties, 2,4-disubstituted pyrrolo[2,3-d]pyrimidine analogs AG114 - AG119 and AG477A were designed and synthesized. Among the analogs tested, AG477A showed an IC50 of 10 ± 0.7 μM in inhibiting tubulin assembly. In drug resistance assays, AG477A showed sub-micromolar inhibitory activity (IC50) against the Pgp over-expressing cell line NCI/ADR-RES (0.89 ± 0.3 μM), as compared with the parental OVCAR-8 cell line (1.2 μM). In addition, AG477A is readily converted to a highly water soluble hydrochloride salt, which marks a significant advantage over existing microtubule targeting agents in clinical use. Structure activity studies show a requirement for the 4-position N-methyl-4-methoxyaniline moiety for antitubulin effects.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3889. doi:1538-7445.AM2012-3889
Abstract 3900: Discovery of FQIT: An imidazo[5,1-f][1,2,4]triazine derived dual IGF-1R/IR inhibitor
Authors: Jin Meizhong , Gokhale Prafulla , Cooke Andy , Foreman Kenneth , Buck Elizabeth , May Earl , Feng Lixing , Bittner Mark , Kadalbajoo Mridula , Landfair Darla , Siu Kam , Stolz Kathryn , Werner Douglas , Laufer Radoslaw , Li An-Hu , Dong Hanqing , Steinig Arno , Kleinberg Andrew , Yao Yan , Pachter Jonathan , Wild Robert , Mulvihill Mark
Published: August 2012

Insulin-like growth factor-1 receptor (IGF-1R) has been recognized as a major target in cancer drug discovery due to its strong implications in various stages of tumorigenesis based on accumulated preclinical data over the years. Recent research on compensatory crosstalk between IGF-1R and insulin receptor (IR) signaling pathways suggests that targeting both receptors is critical to fully blocking the IGF signaling axis. Therefore, inhibition of both receptors is anticipated to result in a more therapeutically beneficial response than targeting IGF-1R alone (e.g. IGF-1R specific antibodies). These findings provided the biological rationale as well as set the foundation for the pursuit and ultimate discovery of OSI-906 (linsitinib), a small molecule dual IGF-1R/IR inhibitor currently in clinical development. As part of OSI's ongoing investment in a small molecule drug discovery platform targeting IGF-1R and IR, a new series of potent and selective imidazo[5,1-f][1,2,4]triazine derived inhibitors of IGF-1R and IR have been identified. Structure-activity relationships and optimization driven by structure-based drug design (SBDD) leading to the discovery of FQIT, a potent, highly selective, well-tolerated and orally bioavailable dual inhibitor of IGF-1R and IR with in vivo efficacy in multiple tumor xenograft models will be discussed.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3900. doi:1538-7445.AM2012-3900
Abstract 3901: Estrogen receptor conformation sensing small molecules as novel anti-cancer agents
Authors: Rankin Bethany , Rajnarayanan Rajendram V. , Koyack Marc
Published: August 2012

Estrogen mediated signaling affects cell proliferation, differentiation and mammary gland function. The human Estrogen Receptor (hER) is present in 70% of breast cancers. Consequently, estrogen mediated hER signaling plays a critical role in breast cancer diagnosis, prognosis, and treatment.Anti-estrogen, Tamoxifen, targets the LBD and transitions the hER from “agonist” conformation into a structurally unique “antagonist” conformation, which is distinctly recognizable by the position of helix XII. Antiestrogen tamoxifen is a known treatment for hER positive breast cancers. However, due to mutations and hormone-independent activation of/in the hER, breast cancers develop resistance to tamoxifen, and resume growth and malignant progression. Estrogen's action at hER is regulated not only by estrogen, but also by other small molecules and coregulators. A closer look at the compliment of coregulators during acquired tamoxifen resistance shows a significant decrease in the corepressor breast cancer susceptibility (BRCA1) protein expression. BRCA1 directly interacts, from amino acids 67-200, with the hER, from 338-379 and 420-495, LBD/AF2 domain. The interface region of BRCA1 containing the binding motif (amino acids 86-93), which specifically contain two essential chemical features, a hydrophobic core and a charged carboxylic acid is necessary for high affinity binding and hER repression. Here we present several small molecules identified based on these pharmacophoric features as well as ER molecular profiles of conformation sensing peptides. Discovery of conformation sensing protein/peptides would open new avenues for drug development and would lead to advanced clinical studies of hER-conformation sensing small molecules as an entirely new class of anticancer therapy especially in tamoxifen-resistant tumors. Acknowledgements: This research was supported via funds from the New York State, National Institutes of Health (5P20MD002725), through the NCMHD -RIMI program (RVR) and NSF LSAMP Bridge to Doctorate grant (BR).

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3901. doi:1538-7445.AM2012-3901
Abstract 3912: The discovery of AZD4547: An orally bioavailable, potent and selective N-(5-Pyrazolyl)benzamide FGFR1-3 inhibitor
Authors: Thomas Andrew P. , Theoclitou Maria-Elena , Buttar David , Ruston Linette , Wrigley Gail , Dennis Mike , Rudge David A. , Coleman Tanya , Smith Robin , Gavine Paul R. , Klinowska Teresa , Mooney Lorraine , Brooks Nigel
Published: August 2012

There is increasing evidence that FGFR signaling plays an important role within human cancer, with members of FGFR family acting as driving oncogenes in a significant number of human tumors. Deregulation of FGFR-signaling has been documented within clinical samples of breast multiple myeloma, bladder, endometrial, gastric, squamous NSCLC and prostate cancers. This dysregulation most frequently occurs through gene amplification, or through genetically altered forms of FGFR proteins. This increasing body of evidence implicating FGFR signaling in cancer has provided rationale for the identification and testing of selective inhibitors of FGFR signaling in the clinic. In this presentation, we describe the progress of our FGFR tyrosine kinase inhibitor programme and report the discovery of N-(5-pyrazolyl)benzamide FGFR inhibitors. Early compounds in this series suffered from poor in vivo pharmacokinetic (PK) properties. The key site of metabolism was identified to be at a basic N-methyl group. This group was shown to be located in the solvent channel of the ATP binding site on binding to FGFR1, and modification could be made without causing major changes to intrinsic binding affinity. However, the first compounds identified with low metabolic clearance also showed a significant reduction in oral bioavailability, due to apparent low permeability and increased efflux potential. The characterization of these PK issues and the discovery of compounds which overcame them, through modulation of pKa, lipophilicity and masking of the polar groups, will be described. Leading compounds showed significant anti-tumor activity in xenograft tumors grown in mice. Detailed characterization of these compounds led to the identification of AZD4547, a potent and selective FGFR tyrosine kinase inhibitor currently in Phase I clinical studies.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3912. doi:1538-7445.AM2012-3912
Abstract 3914: Androgen receptor (AR) degradation is controlled by the co-operation of PMEPA1 and the E3 ubiquitin ligase NEDD4-1
Authors: Li Hua , Dobi Albert , Srivastava Shiv
Published: August 2012

Introduction and Objective: Androgen receptor (AR) plays critical roles in prostate growth and differentiation and prostate tumorigenesis. Our recent investigations have discovered a novel mechanism of AR degradation in CaP cells involving PMEPA1 and NEDD4-1 which is independent of MDM2. Previously we have shown that PMEPA1 is a highly androgen-inducible gene in CaP cells that recruits the NEDD4-1, an E3 ubiquitin ligase, to degrade AR. In an independent study NEDD4-1 has been identified as an E3 ligase controlling the levels of PTEN. We hypothesize that decreased or loss of PMPEA1 expression that is frequently observed in human prostate tumor cells may lead to enhanced AR functions. Elevated AR levels may increase NEDD4-1 and in turn destabilize PTEN in prostate cancer cells. Here, we further investigate the co-operation between PMEPA1 and NEDD4-1 to alter AR and PTEN protein levels. Methods: PMEPA1 and NEDD4-1 were ectopically expressed in androgen responsive LNCaP and VCaP cells by pCMV-XL4 and pcDNA3.1 vectors, respectively. AR, PMEPA1, NEDD4-1, MDM2 and PTEN were silenced by using specific siRNAs. Cell growth was monitored in response modulation of PMEPA1, NEDD4 and AR. Protein levels of AR, PMEPA1, NEDD4-1, PTEN and PSA were assessed by immunoblot assays. Results: Decreases in AR protein levels, inhibited AR signaling and cell growth in response to heterologous expression of NEDD4-1. Consistent with this observation, inhibition of NEDD4-1 by siRNA resulted in elevated AR protein levels, AR signaling and increased cell growth. Reduced cell growth by AR knock-down or by heterologous expression of NEDD4-, were rescued by the knockdown of PMEPA1. It was also shown that AR up-regulates the expression of NEDD4-1 in hormone responsive CaP cells. In VCaP cells, overexpression of NEDD4-1 resulted in decreased PTEN protein levels. Remarkably, PMEPA1 knockdown resulted in elevated AR and NEDD4-1 levels and the downregulation of PTEN protein. Conclusions: NEDD4-1 mediated AR degradation in hormone responsive CaP cells requires PMEPA1. The frequently observed decreased expression of PMEPA1 results in elevated AR protein levels and may facilitate NEDD4-1 mediated degradation of PTEN in CaP. Our findings revealed a complex interplay of the AR, PMEPA1, NEDD4 and PTEN underscoring the impact of the loss of PMEPA1 to increased AR and decreased PTEN, both proteins critical in prostate tumorigenesis. This study is supported by the NIH grant (R01CA106653) to SS.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3914. doi:1538-7445.AM2012-3914
Abstract 3921: Androgens stimulate phosphorylation of ERK and AKT in thyroid cancer cells
Authors: MacEwan Melanie , Swati Ismatun , Xie Suqing , Ardacheva Olena , Iacob Codrin , Shin Edward , Moscatello Augustine , Schantz Stimson , Tiwari Raj K. , Geliebter Jan
Published: August 2012

Thyroid cancer is the most common endocrine malignancy with the papillary variety comprising ∼85% of thyroid neoplasms. While the greatest incidence of PTC is in women of childbearing age, the occurrence of PTC in men and postmenopausal women is associated with increased aggressiveness and poorer clinical outcomes. This gender disparity suggests that while the initiation of PTC is driven by estrogens, its progression may be mediated by androgens. Immunohistochemical analysis of PTC specimens from 5 male and 15 female patients indicated that all expressed androgen receptors (AR). AR mRNA was detected in equivalent amounts in three thyroid cell lines, but the PTC cell lines, BCPAP and TPC-1 expressed significantly more AR protein (4.6 fold and 3.9 fold, respectively) than the “normal” T-antigen transformed thyrocyte cell line, Nthy-ori-1. The treatment of the cell lines with the synthetic androgen, R1881 (10nM) resulted in increased phosphorylation of ERK and AKT in all three cell lines. Nthy expressed 2 fold more pERK when treated with R1881 whereas pAKT, which was previously undetected increased to significantly higher levels. R1881 addition increased pERK and pAKT 3 fold and 2 fold, respectively, in the male, RET/PTC cell line, TPC-1. pERK and pAKT were undetectable in the female, BRAFV600E cell line and were significantly induced by R1881.The known role of ERK and AKT in cellular proliferation, migration and invasion, and the activation of these molecules by androgens, may provide the mechanistic basis for the gender disparity in PTC aggressiveness and clinical outcome between young women and men/postmenopausal women.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3921. doi:1538-7445.AM2012-3921
Abstract 3938: BNip3 limits HIF-1α stabilization and metastasis in a mouse model of breast cancer through effects on mitochondrial integrity and ROS generation
Authors: Tracy Kristin , Chen Hong , Chourasia Aparajita , Macleod Kay F.
Published: August 2012

BNIP3 is a hypoxia-inducible mitochondrial protein that has been implicated in mitochondrial fragmentation and mitophagy. BNIP3 protein is also up-regulated at pre-malignant stages of various different human cancers, including breast cancer and down-regulated later through a variety of mechanisms. To examine the role of BNip3 in tumor progression to metastasis, we crossed mice carrying a targeted deletion in the mouse BNip3 gene to the MMTV-PyMT mouse model of breast cancer. All analyses were performed with mice on a pure FVB/N genetic background. Loss of BNip3 promoted S phase entry that was associated with increased primary tumor growth. There was highly significant increase in the numbers of lung metastases and a reduced latency to metastasis in the MMTV-PyMT;BNip3-/- mice compared to control MMTV-PyMT;BNip3+/+ mice. BNip3 null tumors showed increased invasive properties and nuclear grade at earlier stages, a phenotype that was transplantable to wild-type host mice arguing that the increased invasiveness was intrinsic to BNip3 null tumor cells. BNip3 null tumor cells exhibited abnormal mitochondria, reduced mitochondrial membrane potential, increased ROS levels and increased hypoxia. Their invasive pathology was also associated with increased HIF-1α levels, increased HIF target gene expression and increased CD31-positive blood vessel formation. Quenching ROS limited Hif-1α stabilization, reduced angiogenesis and metastasis in MMTV-PyMT;BNip3-/- mouse tumors to wild-type levels. These results suggest that BNip3 is a metastasis suppressor by virtue of its role in maintaining mitochondrial integrity that mitigates against the metastasis promoting activity of reactive oxygen and hypoxia. Work on this project is supported by NIH/NCI RO1-CA131188 and by Department of Defense BRCP W81XWH-09-1-0587.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3938. doi:1538-7445.AM2012-3938