bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2865: Gemcitabine causes telomere shortening by stabilizing TRF2
Authors: Chih-Hao Su , Ying-Chieh Tsai , Wei-Ping Lee
Published: August 2012

Gemcitabine is an effective anti-cancer agent against sold tumors. The pharmacological mechanism of gemcitabine is known as incorporation into DNA and thereby inhibition of DNA synthesis. Here, we report that gemcitabine treatment not only induced growth arrest and apoptosis in HeLa cells, but also caused telomere shortening without affecting telomerase activity. We performed cycloheximide chase assay to confirm that the half-life of TRF2, a telomere regulator, was prolonged in a dose-pendent manner under the treatment of gemcitabine. In addition, we did in vivo ubiquitination assay and found gemcitabine treatment increased TRF2 protein level by reducing the amount of ubiquitinated TRF2. And, the results of subcellular fractionation and immunostaining showed that the accumulated TRF2 indeed translocates into nucleus. Since TRF2 is a telomeric DNA binding protein, we then made an attempt to determine whether the accumulated TRF2 binds on telomeric DNA. We did chromatin immunoprecipitation and the result revealed that the accumulated TRF2 not only translocates into nucleus but also binds to telomeric DNA. Previous study has shown that XPF-ERCC1, a structure-specific endonuclease and negative mediator of telomere length, interacts with TRF2, also required for TRF2-promoting telomere shortening. To confirm this interaction also exists in HeLa cells under the treatment of gemcitabine, we carried out immunoprecipitation with anti-TRF2 and anti-XPF antibodies and a de-telomeric DNA pull-down assay. Consistently, the results of both assay indicated that increased nuclear TRF2 which was induced by gemcitabine treatment is capable of recruiting more XPF/ERCC1 complex onto telomeric DNA. Besides, the binding studies by experimental manipulation of TRF2 confirmed that the binding ability of XPF-ERCC1 to telomere closely relates to TRF2 and resulted in telomere shortening. Together, these results show that gemcitabine enhances the nuclear accumulation of TRF2 and increased the amount of DNA-bound TRF2, therefore recruiting more XPF/ERCC1 to telomeric DNA and strengthening the nuclease activity of XPF, and resulting in telomere shortening. Since telomere loss is closely related to occurrence of apoptosis, gemcitabine may exert its anti-cancer effect by inducing TRF2 and telomere shortening.

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 2865. doi:1538-7445.AM2012-2865
Abstract 2872: Functional inhibition of HSP90 potentiates the effects of ionizing radiation in colorectal cancer
Authors: Nagaraju Ganji Purnachandra , Wen Jing , El-Rayes Bassel F. , Landry Jerome C. , Park Wungki , Diaz Roberto
Published: August 2012

Purpose/Objectives: Attempts at improving the outcome of colorectal cancer patients by incorporating novel agents have been disappointing indicating that in the majority of patients the therapeutic challenge remains primary resistance to chemoradiotherapy. Major cellular signaling pathways contributing to growth and resistance to chemoradiotherapy in rectal cancer includes the PI3K/AKT/NF-κB pathway. The heat shock protein 90 (HSP90) regulates the activity, turnover, and trafficking of several proteins involved in these pathways of resistance. Inhibition of HSP90 provides a novel mechanism to target these pathways simultaneously. Ganetespib (STA-9090) is a small molecule functional inhibitor of HSP90. We tested the hypothesis that HSP90 inhibition can sensitize colorectal cell lines to the effects of ionizing radiation (XRT). Methods: Immunohistochemical (IHC) analyses of 10 colorectal paired normal and tumor tissue samples from the same 10 patients were stained with antibodies against HSP90, HSP70, and p-AKT. Cell proliferation (XTT), colony formation (clonogenic assay), migration (spheroid), apoptosis (Apoptosis/Necrosis Detection Kit), and Western blot analyses were carried out in HCT-116 and HT-29 colorectal cell lines to determine the effect of ganetespib alone or in combination with XRT. Results: IHC analyses of paired human colorectal normal and tumor samples revealed significant overexpression of HSP90, HSP70, and p-AKT in the tumors when compared to the normal tissues. In both colorectal cell lines, treatment with ganetespib significantly inhibits proliferation in the XTT assay (p<0.001); significantly decreased expression of PI3K and p-AKT, as well as significantly increased levels of HSP70 in Western blot analyses (p<0.05); and inhibited the nuclear translocation (immunocyto studies) and the DNA binding activity (EMSA) of the NF-κB p65 molecules. We found that ganetespib indeed potentiates the effects of XRT in both cell lines: significant decrease in colony formation as determined by the clonogenic assay (p<0.001); increase in the induction of necrosis; and decreased migration. Conclusions: The HSP90 pathway is selectively activated in patient's rectal tumors and is a rational target for drug development. HSP90 inhibition potentiates the effects of ionizing radiation in preclinical models. Evaluating HSP90 inhibitors in rectal cancer is a rational approach that could have a significant impact on the outcome of this common and deadly disease.

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 2872. doi:1538-7445.AM2012-2872
Abstract 2880: Dasatinib induces apoptosis in head and neck squamous cell carcinoma cells through down-regulation of epidermal growth factor receptor (EGFR) in vitro and in vivo
Authors: Lin Yu-Chin , Wu Meng-Hsuan , Wei Tzu-Tang , Chung Shu-Hui , Chen Kuen-Feng , Cheng Ann-Lii , Chen Ching-Chow
Published: August 2012

Background: Head and neck squamous cell carcinoma (HNSCC) is a worldwide disease with aggressive course and dismal outcome. Dasatinib, a bcr-abl inhibitor, has been approved clinically for the treatment of chronic myeloid leukemia and demonstrated to be effective against solid tumors in vitro through Src inhibition. Here, we disclose that dasatinib induced apoptosis of HNSCC cells in vitro and in vivo through down-regulation of EGFR. Methods: HNSCC cells, including Ca9-22, FaDu, HSC3, SAS, SCC-25, and UMSCC1 were treated with dasatinib. Cell viability, apoptosis, and underlying signal transduction were evaluated. Results: Dasatinib exhibited differential sensitivities against HNSCC cells. The growth inhibition and apoptosis were correlated with its inhibition on Akt, Erk, and bcl-2, but irrespective of Src inhibition. Accordingly, we found that down-regulation of epidermal growth factor receptor (EGFR) was a determinant of dasatinib sensitivity. Dasatinib inhibited EGFR through lysosome degradation. Increased EGFR activation by ligand administration rescued cells from dasatinib-induced apoptosis, whereas inhibition of EGFR enhanced its apoptotic effect. Estrogen receptor (ER) was demonstrated to play a role in bcl-2 expression, and dasatinib inhibited ER at pre-translational level. ER worked in concert with EGFR to regulate dasatinib-induced apoptosis. Furthermore, xenograft model showed that dasatinib inhibited HSC3 tumor growth through in vivo down-regulation of EGFR and ER. Conclusion: Down-regulation of EGFR is a novel mechanism responsible for dasatinib-induced apoptosis in HNSCC.

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 2880. doi:1538-7445.AM2012-2880
Abstract 2888: Lipid nanoparticle-mediated delivery of siRNA to extrahepatic tumors
Authors: Davide Joseph P. , Ng Bruce , Mao Xianzhi , Patel Manishkumar , Shi Bin , Hochman Jerome , Cunningham James J. , Sepp-Lorenzino Laura , Tao Weikang
Published: August 2012

While lipid nanoparticles (LNPs) proved to be effective in delivering siRNA to the liver and hepatocellular carcinoma (HCC) via systemic administration, they showed inferior or no efficacy in the extrahepatic tumor models. To understand the underlying mechanism for LNP activity in delivering siRNA to extrahepatic tumors, we determined siRNA biodistribution and gene-silencing activity of the LNP-siRNA nanoparticles containing different percentages of a polyethylene glycol (PEG) lipid, distinct PEG lipids with a short (C-14) or long (C-18) alkyl chain, or an identical composition with different particle sizes in mouse subcutaneous tumor xenograft models derived from either human HCC (Hep3B) or colon cancer (HT29 or HCT116) cells. We show that the LNPs containing C-18 DSA PEG exhibited a higher retention of siRNA in both Hep3B and HT29 tumors than those with C-14 DMA PEG following an intravenous administration. Intriguingly, LNPs containing 2% of DSA or DMA PEG resulted in similar levels (∼50%) of target knockdown in Hep3B tumors while those containing 6% of DSA or DMA PEG caused no target silencing. Also, none of these LNPs reduced target expression in HT29 tumors despite the retention of siRNA in tumor tissues. When LNPs with an identical composition (2% DMG PEG) but different particle sizes (80 nm vs. 120 nm) were tested, we observed a comparable and moderate suppression of target expression in Hep3B tumors, but no target knockdown in HCT116 tumors. This is despite the fact that the expression of low density lipid receptor, which is shown to be involved in mediating LNP efficacy, is detected in HT29 and HCT116 tumors. Finally, intratumor injection of LNPs only induced minimal target silencing in subcutaneous tumors while provoking significant target knockdown in the liver. Together, enhanced tumor retention of siRNA might not be readily translated to target silencing activity in tumors. Other factors such as the efficiency of transfecting tumor cells and the passive targeting effect through opsonization could impact on LNP efficacy in extrahepatic tumors.

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 2888. doi:1538-7445.AM2012-2888
Abstract 2894: Environmentally responsive nanoparticles for the intracellular delivery of RNAi therapeutics into tumor-associated macrophages
Authors: Yu Shann S. , Lau Cheryl M. , Barham Whitney J. , Onishko Halina M. , Nelson Christopher E. , Yull Fiona E. , Duvall Craig L. , Giorgio Todd D.
Published: August 2012

Tumor-associated macrophages (TAMs) represent a potentially promising therapeutic target in cancer because they have been shown to facilitate tumor growth, invasiveness, and metastasis. However, methods to specifically target therapies to TAMs are lacking. To address this problem, we designed and synthesized mannosylated micellar nanoparticles (ManNPs), composed of tri-block co-polymers. The three polymer blocks include (1) an azido-displaying block for functionalization with biomolecules via azide-alkyne ‘click’ chemistry, (2) a cationic block for the condensation of polyanions such as siRNA, and (3) a pH-responsive terpolymer block that facilitates endosomal disruption. This terpolymer is hydrophobic at pH 7.4, allowing these polymers to self-assemble into 25 nm micellar nanoparticles under physiologic conditions. However, they become protonated at lower pH ranges representative of endosomal compartments (5.8 - 6.2), leading to disassembly of the nanoparticles, and increased ability to disrupt endosomal membranes and enable cytoplasmic delivery. This pH-dependent behavior has been validated using a red blood cell hemolysis assay. This environmentally-responsive behavior facilitates improved cytoplasmic delivery of siRNA, access to the intracellular silencing machinery, and consequently, knockdown of target gene expression. Further, we demonstrate that mannosylation of these nanoparticles via ‘click’ chemistry significantly enhances their ability to deliver siRNA into murine bone marrow-derived primary macrophages (BMDMs), relative to control, untargeted nanoparticles. Targeted nanoparticle uptake is mediated specifically through the macrophage mannose receptor (CD206), as validated through competition experiments with free mannose, or by pre-incubating BMDMs with lipopolysaccharide to downregulate CD206 expression. This is particularly important for cancer applications because CD206 is upregulated in tumor-suppressed and non-activated macrophages, enabling more specific targeting of TAMs versus healthy macrophages in other tissues. The ManNPs described here present new opportunities to target TAMs in various cancers, providing an enabling technology for the modification of the immunosuppressive tumor environment by targeting TAM activity. Studies are pending to demonstrate this behavior in an in vivo murine model of metastatic breast cancer.

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 2894. doi:1538-7445.AM2012-2894
Abstract 3017: Oct4B mediates hypoxia induced epithelial mesenchymal trans-differentiation of lung cancer
Authors: Chou Yu-Ting , Chung Chi-Hsiu , Chung Chih-Hung , Wu Cheng-Wen
Published: August 2012

Hypoxia, a reduction of normal oxygen levels in cells or tissues, creates a variety of changes in cell metabolism, eliciting a set of genes involved in tissue development and cancer progression. Though Oct4, a homebox transcription factor, is essential for self-renewal of embryonic stem cells, little is known about the functional role of Oct4 in tumorigenesis. In this study, we discovered that hypoxia induces a short isoform of Oct4, termed as Oct4B, in lung cancer through a HIF-2α dependent pathway. Oct4B induced cell proliferation, anchorage-independent growth, and xenograft tumor formation of lung cancer cells, indicating the oncogenic potential of Oct4B. Moreover, ectopic expression of Oct4B in lung cancer cells induced epithelial mesenchymal tans-differentiation (EMT), promoting cell migration and invasion. We observed that Oct4B bound to Slug promoter upon hypoxia stimulation, promoting EMT as well as invasion of lung cancer cells. Thus, our findings provide a novel mechanism of how Oct4B mediates hypoxia signaling to encourage malignancy of lung cancer.

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 3017. doi:1538-7445.AM2012-3017
Abstract 3019: TGFβ3 is a less potent inducer of TGFβ signaling than TGFβ1 in non-small cell lung cancer cells
Authors: McLean Sarah Elizabeth Anne , Guglielmo Gianni M. Di
Published: August 2012

Transforming growth factor beta (TGFβ) is an essential cytokine for tissue homeostasis. Its signalling pathway is under intense study as it is commonly dysregulated in many cancers, including pancreatic cancer, non-small cell lung cancer, and colorectal cancer. The TGFβ signalling cascade is propagated by the binding of ligand to cell-surface serine-threonine kinase receptors, which leads to the phosphorylation of receptor regulated Smad (R-Smad) proteins. There are three main ligands capable of activating the classical TGFβ pathway: TGFβ1, TGFβ2 and TGFβ3. These ligands show distinct spatial and temporal expression patterns and have non-overlapping functions in vivo. Extensive studies have been conducted on the role of TGFβ1 in the tumour microenvironment, and the role of TGFβ3 has largely been thought to be the same as TGFβ1 though there are few studies to support this claim. Interestingly in the wound microenvironment, TGFβ1 and TGFβ3 have vastly different signalling outcomes: TGFβ1 promotes the formation of a scar while TGFβ3 induces scar-free wound resolution. The objective of the present study was to evaluate the signalling capacity of TGFβ1 and TGFβ3 in non-small cell lung cancer cells. Our studies show that TGFβ3 is less potent at initiating Smad2 phosphorylation than TGFβ1, resulting in reduced transcriptional activity, as assessed by microarray analysis. We also observed that TGFβ3 is less effective than TGFβ1 at altering cell-cell adhesions. In order to assess the differences in signalling potential, we investigated TGFβ receptor engagement at the cell surface using radiolabelled TGFβ ligands. We observed that the ratio of activated receptors in signalling complexes is altered in the presence of TGFβ3, which may lead to a decreased signalling capacity. Future studies will evaluate the capacity of TGFβ3 to induce cancer cell migration and invasion.

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 3019. doi:1538-7445.AM2012-3019
Abstract 3024: Elucidation of WNT signaling pathway players “sFRP-1” and “TCF7L2” as novel potential biomarkers of triple negative breast cancer
Authors: Huelsewig Carolin , Goette Martin , Bernemann Christof , Ruckert Christian , von Wahlde Marie-Kristin , Buchweitz Olaf , Kiesel Ludwig , Pusztai Lajos , Liedtke Cornelia
Published: August 2012

Background: Triple negative breast cancer (TNBC) is characterized by an unfavorable prognosis. While patients do not benefit from endocrine or Her2 targeted therapies except chemotherapy, there is an increased propability of response to taxane-/anthracycline-containing therapy compared to other breast cancers. However, molecular determinants of chemotherapy sensitivity for patients with TNBC specifically remain largely unknown. The WNT signaling cascade has been shown to play an important role in breast cancer pathogenesis. Material and Methods: Differentially expressed genes in triple negative (TN) vs. non-TN breast cancers of patients treated with neoadjuvant taxane-/anthracycline-containing chemotherapy were identified and validated in an independent AffymetrixU133A gene chip dataset. Genes were tested for correlation with relapse-free survival, response to neoadjuvant chemotherapy and Ki67 expression. The effects of siRNA mediated knockdown in the TNBC and non-TNBC cell lines on proliferation, chemo sensitivity invasion competency and gene expression of target genes via quantitative real-time PCR were analyzed. Results: sFRP-1 (secreted frizzled related protein 1) and TCF7L2 (transcription factor-7 like 2) were identified as significantly over expressed genes in TNBC vs. non-TNBC and validated in an independent dataset. While sFRP-1 expression was not associated with relapse-free survival in TNBC, it was significantly correlated with a pCR. Importantly, no correlation to Ki67 expression could be demonstrated. While mRNA knockdown of sFRP-1 in MDA-MB 468 was associated with an increased resistance to paclitaxel, doxorubicin and cisplatin, target gene expression analysis suggest sFRP-1 as a biphasic modulator in breast tissue. TCF7L2 knockdown analysis in the TN cell line MDA MB 231 as well as the non-TN lines MCF7 and SKBR-3 shows an increased cell invasiveness of up to 200 %. Conclusion: We suggest sFRP-1 as predictive biomarker and potential mediator of sensitivity to taxane-, anthracycline and platinum-containing chemotherapy in TNBC in vitro referring to its biphasic WNT modulation. Furthermore TCF7L2 is suggested as a prognostic biomarker in vitro breast cancer cell lines including TN and non-TN cell lines. Our results provide proof of principle that high-throughput gene expression analysis may be exploited as a means to derive at a biological / translational hypothesis that may be supported by basic science.

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 3024. doi:1538-7445.AM2012-3024
Abstract 3027: Dishevelled proteins are highly upregulated in chronic lymphocytic leukemia
Authors: Khan Abdul Salam , Österborg Anders , Moshfegh Ali , Mellstedt Håkan
Published: August 2012

Chronic Lymphocytic Leukemia (CLL) is the most common adult leukemia and is characterized by the progressive accumulation of CD5+ B cells. An important signaling pathway in CLL cells might be Wnt signaling pathways. Wnt signaling pathways include the Wnt/ β-catenin pathway and the non-canonical Wnt pathways. Dishevelled proteins (DVL1, DVL2, and DVL3) are important mediators in Wnt signaling and have been shown in to be expressed many cancer types but their precise role is not clear. The aim of the study was to characterize the expression of DVL1, DVL2 and DVL3 proteins in indolent and progressive CLL and compare to normal white blood cells (PBMC) as well as to assess the expression of other important proteins of the canonical pathway as β-catenin and the non-canonical pathways as GSK-3β, PKC, ERK and AKT respectively. Leukemic cells from indolent (n=9) and progressive (n=9) CLL patients were isolated by Ficoll gradient centrifugation as well as PBMC from healthy donors (n=9). Six cell lines including four CLL cell lines EHEH, CII, I83, 232 B4, and the Jurkat and Lucas cell lines were also used. DVL 1, DVL 2, DVL 3 were analyzed by Western blot and densitometric calculations as well as PKC, GSK-3β, AKT and ERK using antibodies against the total protein and the phosphorylated protein respectively. Overexpression of DVL1, DVL 2, and DVL3 was seen in all indolent and progressive CLL patients and the six cell lines. There was a significantly higher expression of DVL 1 and DVL 3 in indolent compared to progressive CLL patients (p<0.01 and p<0.01 respectively). In contrast to DVL 1 and DVL 3, DVL 2 was highly expressed in indolent as compared to progressive CLL patients. ≤ -catenin followed the same expression pattern as DVL2 being highly expressed in indolent CLL as compared to progressive CLL patients (p<0.001). There was no significant difference in the degree phosphorylation of PKC and GSK-3β between indolent CLL, progressive CLL and healthy donors.The degree of AKT and ERK phosphorylation was significantly higher in indolent than in progressive CLL patients (p<0.001 and p<0.01 respectively). In summary DVL1, DVL2 and DVL3 are highly expressed in CLL patients and CLL cell lines but their expression in normal PBMC is almost negligible but expression of other canonical and non-canonical pathways proteins are seen both in CLL and healthy donors. DVL1, DVL2 and DVL3 may have a key role in the transduction of Wnt mediated signals in CLL.

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 3027. doi:1538-7445.AM2012-3027
Abstract 3029: Biochemical evidence towards the existence of an oncogenic Hsp90 complex
Authors: Rodina Anna , Moulick Kamalika , Ahn James , Zong Hongliang , Cerchietti Leandro , DaGama Erica Gomes , Caldas-Lopes Eloisi , Beebe Kristin , Perna Fabiana , Hatzi Katerina , Vu Ly , Zhao Xinyang , Zatorska Danuta , Taldone Tony , Smith-Jones Peter , Alpaugh Mary , Gross Steven , Pillarsetty Nagavarakishore , Ku Thomas , Lewis Jason , Larson Steven , Levine Ross , Erdjument-Bromage Hediye , Guzman Monica , Nimer Stephen , Melnick Ari , Neckers Len , Chiosis Gabriela
Published: August 2012

To maintain homeostasis, cells employ intricate molecular machineries comprised of thousands of proteins programmed to execute well-defined functions. Dysregulation of these pathways, through protein mis-expression or mutation, provides biological advantages that confer the malignant phenotype. At the molecular level, this requires cells to invest energy in maintaining the stability and function of these proteins, and for this reason cancer cells co-opt molecular chaperones, including Hsp90. Hsp90 is recognized to play important roles in maintaining the transformed phenotype - the chaperone and its associated co-chaperones assist in the correct folding of cellular proteins, collectively referred to as “client proteins,” many of which are effectors of signal transduction pathways controlling cell growth, differentiation, the DNA damage response, and cell survival. Tumor cell addiction to these proteins (i.e. through mutations, aberrant expression, improper cellular translocation, etc) thus makes them critically reliant on Hsp90. While Hsp90 is expressed in all cells and tissues, it was shown that tumors preferentially contain Hsp90 that is in a higher order multi-chaperone complex with high affinity for certain Hsp90 inhibitors, while normal tissues harbor a latent, uncomplexed Hsp90 that has low affinity for these inhibitors. We here extend this model and propose that Hsp90 forms biochemically distinct complexes in cancer cells. In this view, a major fraction of cancer cell Hsp90 retains “house keeping” chaperone functions similar to normal cells, whereas a functionally distinct Hsp90 pool enriched or expanded in cancer cells specifically interacts with oncogenic proteins required to maintain tumor cell survival. Perhaps this Hsp90 fraction represents a cell stress specific form of chaperone complex that is expanded and constitutively maintained in the tumor cell context. Our data suggest that it may execute functions necessary to maintain the malignant phenotype. One such role is to regulate the folding of mutated (i.e. mB-Raf) or chimeric proteins (i.e. Bcr-Abl). We here also present experimental evidence for an additional role; that is, to facilitate scaffolding and complex formation of molecules involved in aberrantly activated signaling complexes.

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 3029. doi:1538-7445.AM2012-3029
Abstract 3032: Role of ruffles and filopodia in adhesion gradient sensing
Authors: Heckman Carol A. , Amarachintha Surya P.
Published: August 2012

Filopodia play a sensory role in directing motility during embryonic development and axon pathfinding. They also show a low prevalence in cancer cells. Here, we determine whether cells from a rat tracheal epithelial line in vitro use filopodia to sense adhesive gradients. Cells exhibit haptotaxis (movement toward a more adhesive surface) when placed on a metal-glass gradient. Tantalum (Ta) and platinum (Pt) gradients were created on glass slides and high (H), middle (M), and low (L) positions defined along the gradient. Cell counts in randomly selected fields confirmed that the cells recognized the gradient. To determine whether the prevalence of protrusions differed at the H, M, and L locations, we measured the values of latent factors 4 (filopodia), 5, and 7. Factor 4 values were high at H and significantly lower at M and L (p<0.05). Cells’ ability to form large protrusions, represented by factors 5 (lamellar distribution) and 7 (nascent neurites), was uniform across the Ta gradient. To detect the cells’ ability to interpret directional cues, we distinguished the cell's top (T) side, i.e. the side oriented toward H location, from the bottom (B) side. Factor 4 values at H-T exceed those at M-B and L-T (Table 1). Trend analysis confirmed a decrease in factor 4 over the gradient with significance of p<0.001 (Ta) and p<0.023 (Pt). On Pt only, factor 5 increased (p=0.000) as the metal content of the substrate declined. Ruffling activity is generally inverse to filopodia but positively related to factor 5 (Heckman et al., doi:10.1016/j.cellsig.2011.08.023). On haptotactic substrates, the former relationship was observed by correlation analysis but was restricted to the L location on the gradient. This suggests that poor adhesion may help to convert mechanisms of filopodia formation to ruffle formation. The above results suggest that sensing depends on net filopodia stabilization at the more adhesive H location. We conclude that filopodia respond to the gradient and that regulation of the sensing and signaling mechanisms can now be evaluated.

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 3032. doi:1538-7445.AM2012-3032
Abstract 3034: Hsp90-dependent stabilization of active NPM-ALK kinase: Structural insights by mutagenesis of the ALK catalytic domain
Authors: Bonvini Paolo , Mussolin Lara , Cozza Giorgio , Moro Stefano , Rosolen Angelo
Published: August 2012

Background. Among the posttranslational modifications occurring in eukaryotic cells, phosphorylation is the most critical for proteins, as phosphate-mediated conformational switching of catalytic and non-catalytic motifs impacts expression and activity through changes in structure stability. Activation by phosphorylation is the rule for protein kinases, including oncogenic protein tyrosine kinase NPM-ALK (nucleophosmin-anaplastic lymphoma kinase) in Anaplastic Large Cell Lymphomas, though it renders the kinase prone to degradation. For several of them, however, the maintenance of functional stability is provided by the molecular chaperone Hsp90, through the recognition of specific motifs within the catalytic domain. Objectives. Herein, we determined whether NPM-ALK kinase activity facilitates Hsp90 binding, and whether structural motifs within NPM-ALK catalytic domain favor or impair Hsp90 interaction. Design/Methods. To test this hypothesis we prepared a series of N- and C-terminal deletion mutants of wild-type NPM-ALK, as well as inactive point mutants by site-directed mutagenesis of the NPM-ALK ATP-binding site and activation loop. Results. Using NPM-ALK constructs in COS7 and 293T cells, we showed that Hsp90 strongly interacts with wild-type NPM-ALK, but fails to co-adsorb with inactive point mutants and with deletion mutants lacking the entire kinase domain. By comparison, binding to Hsp90 family members Hsc70 and Hsp70 is independent of NPM-ALK activity, suggesting a greater dependence of active NPM-ALK on Hsp90 function. Accordingly, phosphorylated NPM-ALK associated with Hsp90 and co-chaperone cdc37, and pharmacological inhibition of NPM-ALK prevented the interaction of NPM-ALK with both proteins. Of note, mutants unmasking the αC-helix motif in the N-terminal lobe of the catalytic domain maintained the ability to bind Hsp90, whereas mutants lacking αC-helix did not. Conclusions. Our findings, therefore, suggest a model in which, in addition to a role in folding nascent NPM-ALK, Hsp90 provides an “ongoing folding support” to activated NPM-ALK kinase, by protecting self-phosphorylated NPM-ALK from ubiquitination through recognition of specific unstable motifs within its catalytic domain.

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 3034. doi:1538-7445.AM2012-3034
Abstract 3035: Bone morphogenetic proteins signal through Smad2 and Smad3 to regulate cell migration and proliferation
Authors: Holtzhausen Alisha , How Tam , Gersh Bradley C. , Blobe Gerard C.
Published: August 2012

The TGF-β superfamily consists of a large number of growth factors, including transforming growth factor-β (TGF-β) and bone morphogenetic proteins (BMPs) that regulate cellular proliferation, differentiation, invasion, migration and apoptosis. TGF-β activates the transcription factors Smad2/3, as well as Smad1/5/8, while BMP stimulation results in the activation of Smad1/5/8. Unexpectedly, we have demonstrated that BMP family members, including BMP2, 3, 4, 7, 9 and GDF5 can also induce Smad2/3 phosphorylation and downstream gene transcription in ovarian, pancreatic and breast cancer cell lines. BMP2-induced Smad2 phosphorylation can be attenuated by expression of dominant negative TβRII or shRNA-mediated knockdown of TβRII, while BMP2-induced Smad3 phosphorylation can be attenuated by expression of dominant negative BMPRII or shRNA-mediated knockdown of BMPRII, suggesting different mechanisms of BMP-mediated activation of Smad2 and Smad3. BMP2-induced Smad1, Smad2 and Smad3 phosphorylation increase cell invasion, migration and proliferation, respectively. BMP stimulated Smad2/3 phosphorylation suggests that BMP-mediated effects on cancer progression may be regulated, in part, through these novel signaling pathways. Current studies are focused on further elucidating the role of BMP-induced Smad2 and Smad3 phosphorylation on human cancer progression.

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 3035. doi:1538-7445.AM2012-3035
Abstract 3036: Interaction of proliferation cell nuclear antigen (PCNA) with c-Abl in cell proliferation and response to DNA damages in breast cancer
Authors: Zhao Hua Jun , Ho Po-Chun , Lo Yuan-Hung , Espejo Alexsandra , Bedford Mark T. , Hung Mien-Chie , Wang Shao-Chun
Published: August 2012

Cell proliferation in primary and metastatic tumors is a fundamental characteristic of advanced breast cancer. Further understanding of the mechanism underlying enhanced cell growth will be important in identifying novel prognostic markers and therapeutic targets. Here we demonstrated that tyrosine phosphorylation of the proliferating cell nuclear antigen (PCNA) is a critical event in growth regulation of breast cancer cells. We found that phosphorylation of PCNA at tyrosine 211 (Y211) enhanced its association with the non-receptor tyrosine kinase c-Abl. We further demonstrated that c-Abl facilitates chromatin association of PCNA and is required for nuclear foci formation of PCNA in cells stressed by DNA damage as well as in unperturbed cells. Targeting Y211 phosphorylation of PCNA with a cell-permeable peptide inhibited the phosphorylation and reduced the PCNA-Abl interaction. These results show that PCNA signal transduction has an important impact on the growth regulation of 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 3036. doi:1538-7445.AM2012-3036
Abstract 3037: Cyr61/CCN1 regulates SHh through Notch signaling in pancreatic cancer cells
Authors: Haque Inamul , Majumder Monami , Banerjee Sushanta K. , Mehta Smita , Banerjee Snigdha
Published: August 2012

Cyr61 (cysteine-rich 61)/CCN1, a member of the CCN family of growth factors that includes CTGF, NOV, WISP-1, WISP-2 and WISP-3, is associated with the development of various cancers. Our recent studies found Cyr61/CCN1 play a critical role in pancreatic carcinogenesis through the induction of EMT and stemness. Cyr61 mRNA and protein were detected in the early precursor lesions and their expression intensified with disease progression. However, the mode of action of CCN1 in the pancreatic carcinogenesis is unknown. In this study, we have shown that CCN1 regulates the expression of Sonic hedgehog (SHh), a lipid-modified secreted signaling protein, plays a critical role in pancreatic cancer development from Pan1N lesions to invasive growth of the disease. Moreover, we also found that SHh regulation by CCN1in pancreatic cancer cells is mediated through the regulation Notch1 signaling pathway. These extensive studies proposed that targeting Cyr61/CCN1 can provide a new treatment option for patients with pancreatic cancer as blocking CCN1 simultaneously two critical pathways (i.e., SHh and Notch1) associated with the development of the disease as well as drug resistence. (THIS WORK IS SUPPORTED BY VA MERIT GRANT TO SB).

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 3037. doi:1538-7445.AM2012-3037
Abstract 304: Proinflammatory secretory chemokine CXCL8 regulates intercellular cross-talk in the breast cancer tumor microenvironment
Authors: Jussim Casey , George Andrea L. , Rajoria Shilpi , Suriano Robert , Shanmugam Arulkumaran , Mittelman Abraham , Tiwari Raj K.
Published: August 2012

The cellular composition of the tumor microenvironment (TME) and soluble effector molecules mediating intercellular cross-talk are important therapeutic and preventive targets for breast cancer. Chemokines released within the TME work in a synergistic and reciprocal manner to induce malignant progression of tumor cells. CXCL8, a proinflammatory CXC chemokine shown to be released within the TME, can act as a chemoattractant, angiogenic factor, and inflammatory factor via cellular receptors CXCR1 and CXCR2. However, the specific role of CXCL8 in the signaling cascades within the tumor microenvironment has yet to be fully classified and warrants further investigation. Treatment of human breast cancer cell lines MCF-7, MDA-MB-231, and T47D with 50nM exogenous CXCL8 enhanced the expression of CXCR2. The acquisition of chemokine receptors by tumor cells, in this case CXCR2, can mediate phenotypic changes that enhance migratory and invasive potential. Treatment of these human breast cancer cell lines with 50nM exogenous CXCL8 resulted in phenotypic morphology changes characteristic of an epithelial-to-mesenchymal transition (EMT), a phenomenon commonly exhibited by metastatic tumor cells. We validated this phenomenon with 50nM exogenous CXCL8 treatment, which led to enhanced expression of both Slug (Snail2) and Twist in all three human breast cancer cell lines. Furthermore, we observed that treatment with 50nM exogenous CXCL8 promoted the migration and invasion of these breast cancer cell lines in an in vitro modified Boyden chamber assay. These results suggest that CXCL2 secreted from cells within the TME act to induce characteristic changes in human breast cancer cells, propagating the transition to more aggressive tumors. Tumor-associated macrophages (TAMs) are an important component of the heterogeneous population of cells that make up the TME. Breast cancer cells exposed to conditioned media from human monocytic cell lines THP-1 and U937, previously differentiated with 12-O-tetradecanoylphorbol-13-acetate (TPA), exhibited distinct morphology changes characteristic of EMT and enhanced migratory and invasive ability, similar to exogenous CXCL8 treatment. Together, our results suggest that CXCL8 can have profound implications in determining the fate of tumor cells by engaging in extensive cross-talk within the TME, thus providing a potential target for the treatment and prevention of breast cancer tumor progression and metastasis.

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 304. doi:1538-7445.AM2012-304
Abstract 3041: Macrophage inhibitory cytokine-1 stimulates proliferation of human umbilical vein endothelial cells by up-regulating Cyclins D1 and E through the PI3K/Akt- and ERK-dependent AP-1 and E2F activation signaling pathways
Authors: Jin Young-June , Lee Jeong-Hyung , Kim Young-Myeong , Kim Keun-Cheol , Lee Hansoo
Published: August 2012

Macrophage inhibitory cytokine-1 (MIC-1) is highly associated with malignant human cancers and has been suggested to be involved in tumor angiogenesis. In the present study, we examined the effect of MIC-1 on proliferation of endothelial cells. MIC-1 treatment accelerated progression of the G1 stage in cell cycle of human umbilical vein endothelial cells, together with increased cell proliferation. MIC-1 augmented the levels of cyclin D1 and E without affecting the levels of CDK inhibitors, thereby increasing activity of CDK, which led to Rb phosphorylation and subsequent E2F-1 nuclear translocation. MIC-1-induced expression of cyclins D1 and E was found to be mediated by AP-1 and E2F-1 transcription factors, and among the AP-1 members, c-Jun and JunD appeared to participate in MIC-1-mediated up-regulation of cyclin D1. Additionally, PI3K/Akt and ERK were found to mediate MIC-1-induced cyclin D1 expression. Importantly, lung endothelial cells of MIC-1 transgenic mouse exhibited increased cell proliferation rate and cyclin D1 and E levels as compared to the wild-type mouse counterparts. These results suggest that MIC-1 secreted from cancer cells stimulates endothelial cell proliferation by up-regulating of the G1 cyclins through the PI3K/Akt- and ERK-dependent AP-1 and E2F activation signaling pathways, potentially leading to the promotion of tumor angiogenesis.

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 3041. doi:1538-7445.AM2012-3041
Abstract 305: Modulation of CXCL-8 expression in human melanoma cells regulates tumor growth, angiogenesis, invasion and metastasis
Authors: Wu Sheng , Singh Seema , Kindle Scott , Varney Michelle L. , Singh Rakesh K.
Published: August 2012

CXCL-8, a chemokine secreted by melanoma and stromal cells, serves as a growth and angiogenic factor for melanoma progression. This study evaluated how modulation of CXCL-8 levels in melanoma cell lines with different tumorigenic and metastatic potentials affected multiple tumor phenotypes. A375P cells (CXCL-8 low expressor) were stably transfected with a CXCL-8 mammalian expression vector to overexpress CXCL-8, whereas A375SM cells (CXCL-8 high expressor) were transfected with a CXCL-8 antisense expression vector to suppress CXCL-8 expression. Subsequent cell proliferation, migration, invasion, and soft-agar colony formation were analyzed and in vivo tumor growth and metastasis were evaluated using mouse xenograft models. Our data demonstrate that over-expression of CXCL-8 significantly enhanced primary tumor growth and lung metastasis, accompanied by increased microvessel density in vivo, as compared to vector control transfected cells. We also observed increased colonogenic ability, growth and invasive potential of CXCL-8 over-expressing cells in vitro. Knock-down of CXCL-8 using an antisense vector resulted in increased cell death and reduced tumor growth relative to control. Taken together, these data confirm that CXCL-8 expression plays a critical role in regulating multiple cellular phenotypes associated with melanoma growth and metastasis.

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 305. doi:1538-7445.AM2012-305
Abstract 3050: Inducing apoptosis in mantle cell lymphoma using inhibitors of cyclin-dependent kinases and the Bcl-2 inhibitor ABT-737
Authors: Lo Wann-Cheng , LeBrun David P. , Greer Peter A.
Published: August 2012

Mantle cell lymphoma (MCL) is a cyclin D1 over-expressing B cell lymphoma which displays unregulated cell cycle progression driven by increased cyclin-dependent kinase (CDK) activity. We are investigating selective small molecule CDK inhibitors as potential therapeutics in MCL. AZD5438, flavopiridol, PD-0332991 and SNS-032 have distinct efficacies against different groups of CDKs. We hypothesize that inhibiting CDK-7 and -9 will be more effective in causing cell death in MCL than inhibiting CDK-1,-2,-4 and- 6. In Jeko-1 cells, AZD5438 and flavopiridol displayed time- and dose-dependent inhibition of cell proliferation, inhibition of the phosphorylation of CDK substrates, and G1 cell cycle arrest; while PD-0332991 showed a strong cytostatic effect by inhibiting cell proliferation and arresting cells at the G1 phase, but not inducing apoptosis to the same extent as flavopiridol and AZD5438. In addition, AZD5438, flavopiridol and SNS-032 induced caspase-3 activation and down regulation of Mcl-1, but no apoptotic effect was shown in PD-0332991 treated cells. These differential effects may be associated with essential survival functions conferred by genes regulated by CDK7/9 including the pro-survival protein Mcl-1. However, there were no apoptotic effects observed with any of these CDK inhibitors in Granta-519 cells, which may due to high Bcl-2 levels. We are now exploring combinations of CDK7/9 inhibitors with a small molecule inhibitor of Bcl-2 function. We expect CDK7/9 inhibition will reduce Mcl-1 expression at the transcriptional level and thereby sensitize both Jeko-1 and Granta-519 cells to Bcl-2 inhibition. We will also investigate Mcl-1 and Bcl-2 levels in clinical samples to see if there is a correlation with outcomes. Our expectation is that Mcl-1/Bcl-2 levels may ultimately become a useful prognostic and predictive biomarker in MCL.

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 3050. doi:1538-7445.AM2012-3050