bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3125: EGFRvIII translocates to the nucleus and regulates gene transcription with STAT5
Authors: Latha Khatri , Chumbalkar Vaibhav , Li Ming , Hwang YeoHyeon , Gururaj Anupama , Maywald Rebecca , Dakeng Sumana , Dao Lixia , Baggerly Keith , Sawaya Raymond , Cavenee Webster , Furnaru Frank , Bögler Oliver
Published: February 2011

Dysregulation of receptor tyrosine kinases is a major contributor to cancer, including glioblastoma, where amplification and mutation of epidermal growth factor receptor (EGFR) is common. The most common mutation is in-frame deletion of exons 2-7, known as EGFRvIII, EGFR* or ΔEGFR. EGFRvIII confers enhanced tumorigenicity on glioma cells in vivo, reducing apoptosis and increasing proliferation. EGFRvIII signals constitutively and at approximately 5-to-10-fold lower intensity than wild-type.

We are investigating the pathways that are preferentially activated by EGFRvIII, using a phosphotyrosine-directed, mass spectrometry-based shotgun phosphoproteomics approach in two different cell lines, LNZ308 (PTEN null) and LN428 (PTEN wt). We identified 433 proteins with 772 peptides out of which 249 proteins corresponding to 354 peptides showed tyrosine phosphorylation. Statistical analysis revealed several signals that were prevalent in EGFRvIII expressing glioma cells, including phosphorylation of STAT5, Gab1 and MIG6.

Identification of STAT5 as a target of EGFRvIII prompted an investigation of whether EGFRvIII is active in the nucleus. We have identified EGFRvIII associated with phosphorylated STAT5 on chromatin, and capable of activating the expression of STAT5 target genes, including Aurora A kinase, which positively regulate glioblastoma malignancy. STAT5 promotes expression of the EGFRvIII target gene Bcl-XL, and knockdown of STAT5 reduces the transformation induced by EGFRvIII. Furthermore, we have mutated signals in EGFRvIII that regulate nuclear localization, and are examining the impact of these on its ability to promote tumor growth.

In a related study we have used artificial receptor dimerization, mediated by technology from Ariad Pharmaceuticals, to enhance the EGFRvIII signal. A chimeric version of EGFRvIII was created, in which the receptor was fused N-terminally with two FKBP-F36V domains that could be activated by the chemical inducer of dimerization, AP20187. This has resulted in a more oncogenic form of EGFRvIII that emits a stronger signal, and we are using this to deepen our phosphoproteomics analysis to seek additional preferential targets of EGFRvIII action, which will be reported.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3125.
Abstract 3133: In vitro modeling of human pancreatic duct epithelial cell transformation
Authors: Leung Lisa H. , Radulovich Nikolina , Tsao Ming-Sound
Published: February 2011

Background: Pancreatic cancer is the fifth most common cause of cancer death in North America and has an overall 5-year survival rate of less than 5%. Over 90% of tumours are ductal carcinoma (PDAC), which putatively arise from the pancreatic ducts. In PDAC, KRAS oncogene activation occurs in approximately 90% of cases, while SMAD4 tumour suppressor gene inactivation occurs in around 55% of cases. We hypothesize that introduction of multiple genetic aberrations into pancreatic epithelial cells derived from normal ducts may provide a dynamic model to study pancreatic duct cell carcinogenesis.

Materials and Methods: We have isolated another cell line from the H6c7 immortalized near normal pancreatic duct epithelial (HPDE) cell line called TβR. TβR cells were generated by growing the H6c7 cells in progressively increasing concentrations of TGF-β and are resistant to growth inhibition by TGFβ. Overexpression of KRASG12V in TβR gave rise to TβR-KRAS cells.

Results: Spectral karyotypic analysis of TβR cells revealed paradiploidy but with the loss of one copy of chromosome 18, on which the Smad4 gene resides. Q-PCR and Western blot confirmed the lost of Smad4 expression in this cell line. TβR cells demonstrated increased invasiveness as compared to the parental H6c7 cell line, and this is further enhanced in TβR-KRAS cells. The TGF-β-Smad4 signaling pathway was disrupted in both the TβR and TβR-KRAS cell lines. This resulted in the loss of responsiveness to TGF-β and the repressed expression of Smad4 responsive genes. The subcutaneous injection of the TβR-KRAS induced in tumor growth in SCID mice.

Conclusion: The complete loss of Smad4 expression cooperates with KRASG12V oncogene to induce tumorigenicity in H6c7 cells. This provides a dynamic in vitro model to study the gene expression changes that characterize the key molecular events that occur during pancreatic ductal carcinogenesis. (Supported by the Canadian Institutes of Health Research grant MOP-49585)

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3133.
Abstract 3148: Translational control of lung cancer: eIF4E over-expression is not sufficient to confer tumorigenicity to human bronchial epithelial cells
Authors: McCauley Joel , Jacobson Blake , Bitterman Peter , Kratzke Robert , D'Cunha Jonathan
Published: February 2011

INTRODUCTION: Lung cancer is the leading source of cancer mortality in North America. There is an incomplete understanding of lung cancer tumorigenesis and new therapeutic targets are clearly needed. The role of 5’ cap-mediated translation in the control of cell growth and proliferation has become increasingly evident over the last decade. Enhanced cap-mediated translation has been implicated in multiple human malignancies including non-small cell lung cancer (NSCLC). Our group has shown that increased levels of eukaryotic initiation factor 4E (eIF4E) have been demonstrated in NSCLC and in vitro studies have shown that ectopic expression of eIF4E primary mammary epithelial cells enables clonal expansion and anchorage independent growth. Thus, our goal was to determine if over-expression of eIF4E would confer tumorgenicity to both normal human bronchial epithelial cells (NHBE), hTERT/Cdk4-immortalized human bronchial epithelial cells (HBECs), and SV40-transformed HBECs.

METHODS: Over-expression of eIF4E was stably induced by lentiviral transduction in NHBE, HBECs, SV40-transformed HBECs, and HBECs expressing lung cancer relevant oncogenes. Western blot was used to confirm eIF4E expression. Enhanced cap-mediated translation was confirmed by a cap-binding assay and soft agar assays were performed to determine anchorage independent growth. The stably transfected cells were injected into nude mice and evaluated for tumor formation.

RESULTS: Over-expression of eIF4E was accomplished by lentiviral transduction and shown to increase 5’ cap binding by cap-binding assay. Over-expression of eIF4E did not induce loss of cellular senescence in NHBEs. HBECs stably transformed with eIF4E did not exhibit anchorage independent growth. The over-expression of eIF4E in conjunction with p53 knockdown, K-RASV12, and c-myc did not confer in vitro evidence of tumorigenesis or lead to tumor formation in a xenograft model. Finally, the over-expression of eIF4E in SV40-transformed HBECs did not confer tumorigenesis in vitro or in vivo.

CONCLUSIONS: Over-expression of eIF4E in NHBEs, hTERT/Cdk-immortalized HBECs, and SV40-immortalized HBECs was not sufficient to induce tumorigenesis. Further, the addition of several key oncogenes did not drive transformation. Additional investigations are warranted to evaluate the important translational regulators in NSCLC tumorigenesis. The resistance to tumor formation seen in this study may provide important insight into the regulatory mechanisms present in bronchial epithelial cells routinely exposed to environmental carcinogens.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3148.
Abstract 3152: Sumoylation of eIF4E activates mRNA translation
Authors: Hu Jing , Xu Xiang , Vatsyayan Jaya , Gao Chenxi
Published: February 2011

Eukaryotic translation initiation factor 4E (eIF4E) is the cap-binding protein that binds the 5’ cap structure of cellular mRNAs. Despite the obligatory role of eIF4E in cap-dependent mRNA translation, how the translation activity of eIF4E is controlled remains largely undefined. Here we report that mammalian eIF4E is regulated by SUMO-1 (small ubiquitin-related modifier-1) conjugation. eIF4E is sumoylated on lysines 36, 49, 162, 206 and 212. Using eIF4E sumo-deficient mutant as a tool, we find that eIF4E sumoylation disrupts the interaction of 4IF4E with its inhibitor 4E-BP1 (eIF4E-binding protein 1) and promotes the eIF4E/eIF4G association. Further, downregulation of eIF4E sumoylation inhibits eIF4E-dependent protein translation of a subset of genes that are critical for cell proliferation and preventing apoptosis, and abrogates the oncogenic and anti-apoptotic functions associated with eIF4E. These data indicates that sumoylation represents a novel fundamental regulation mechanism of protein synthesis. Our findings further suggest that eIF4E sumoylation may be important in promoting human cancers.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3152.
Abstract 3164: Posttranscriptional regulation of opiate tolerance by OPRM1 splice variants following morphine/fig (Ficus carica) leaf extract treatment of human neuroblastoma (SH-SY5Y) cells
Authors: Lightbourn Alrena V. , Goodman Carl B. , Zhu Zhi-Ping
Published: February 2011

Aberrant splicing events have been implicated in a growing number of diseases, including cancer. The pharmacological significance of newly identified mu opioid receptor (MOR-1) alternative splice variants has not been characterized relative to drug response mechanisms and may inform the issue of morphine tolerance. Among the 70-90% of cancer patients requiring individualized opioid therapy for intense chronic pain, the response to prototypical opiates like morphine is highly variable, necessitating dose escalation with an increased risk of developing tolerance. It has been suggested that chronic morphine exposure has little, if any, effect on MOR-1 regulation. If this is true, then chronic exposure should also have no effect on variant forms of receptor. We therefore tested the hypothesis that, following chronic exposure to morphine and/or fig leaf extract, MOR-1 variants would be differentially regulated as evidenced by altered relative mRNA expression levels. Retinoic-acid differentiated human neuroblastoma (SH-SY5Y) cells were treated with morphine (10 µM), fig leaf extract (3 µL/30 mL media), or both for 48 hours before analysis by quantitative real-time polymerase chain reaction (qRT-PCR) using the Bio-Rad iCycler/MyiQ™. MOR-1 variant forms identified by analysis included: MOR1A, MOR1-B1, MOR1-B2, MOR1-B3, MOR1-B5, MOR1-K1. MOR1B4 expression was negligible. Variant mRNA expression was differentially regulated in 92% of the samples treated with fig leaf extracts from 7 cultivars (Green Ishia, Brown Turkey, Mission, Alma, Giant Celeste, Nero, Hollier). Fig administered alone up-regulated MOR-1 splice variant expression (1.6 to 16.9-fold), but these levels were suppressed in combination with morphine (0.2 to 0.8-fold). Also in combined treatment groups, MOR1-K1 and MOR1-B2 mRNA levels consistently up-regulated or down-regulated together. Morphine given alone down-regulated variant expression (0.2 to 0.7-fold). Closer inspection of MOR-1 alternative splice variants under experimental conditions appear to disqualify the assumption that MOR-1 receptor expression remains unchanged under chronic exposure conditions. The results of this study confirm our hypothesis that MOR-1 splice variants are differentially regulated following chronic exposure to morphine and other agents. Further examination of the functional role of MOR-1B2 and MOR1-K1 variants may contribute to our understanding of the mechanistic basis of morphine tolerance and may give clues as to the therapeutic benefit of targeting transcriptional isoforms the receptor for cancer pain relief.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3164.
Abstract 3184: TGF-β1 signaling inhibits proliferation but promotes motility with progression to castrate-resistant prostate cancer
Authors: Miles Fayth L. , Sikes Robert A.
Published: February 2011

Transforming growth factor-beta1 (TGF-B1) is a tumor suppressor in normal and early stage prostate cancer (PCa) cells, but exerts pro-tumorigenic effects during progression to metastatic disease, and correlates with poor prognosis. However, the mechanisms whereby TGF-B1 induces pro-metastatic action are uknown. Previously it was reported that the LNCaP cell line was insensitive to TGF-B-mediated growth suppression. Thus we sought to examine the TGF-B1-induced proliferative response of increasingly metastatic, lineage-related LNCaP sublines.

Under reduced serum conditions we show that TGF-B1 inhibits cell proliferation profoundly and promotes apoptosis in castrate-insensitive LNCaP sublines, whereas inhibition of cell proliferation is marginal in LNCaP cells. Growth inhibitory effects and apoptosis are mediated by active TGF-B receptors, TBRII and TBRI, and the downstream Smad2/3 effectors. Knock down of TBRII with a dominant negative construct, as well as inhibition of kinase activity abrogrates these effects. Additionally, we show that TGF-B enhances cytoskeletal rearrangements and increases motility on selected extracellular matrices in metastatic LNCaP sublines.

TGF-B signaling in castrate-resistant PCa cell lines is more robust than in hormonally sensitive clones and leads to selective activation of cell motilty that influences PCa cell metastatic behavior with potential consequences for bone colonization.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3184.
Abstract 3190: Heparanase modulates Shh and Wnt3a signaling in human medulloblastoma cells
Authors: Ridgway Lon D. , Wetzel Michael E. , Marchetti Dario
Published: February 2011

The pathogenesis of medulloblastoma (MB), the most common and aggressive brain tumor in children, is poorly understood. MB tumors are characterized by a massive expansion of external granule layer cells of the cerebellum in response to Purkinje neurons secreted factors such as Sonic hedgehog (Shh) and Wnt3a. Understanding the modulation of Shh/Wnt signaling is critical to developing new treatments for medulloblastoma. Shh and Wnt3a are involved in the induction of MB cell proliferation, and bind heparan sulfate glycosaminoglycan chains (HS-GAG). HS-GAG are components of syndecans as cell-surface HS proteoglycans (HSPG), important co-receptors involved in the control of signaling by extracellular matrix - based ligands. We hypothesized that Shh/Wnt3a - induced MB cell signaling is mediated by syndecans, altering syndecan carboxy terminal (CT) - associated molecules and downstream targets. We also hypothesized that these events are regulated by heparanase (HPSE), the only functional mammalian endoglycosidase cleaving HS-GAG at intrachain site and widely implicated in cancer metastasis. We compared regulation of Shh/Wnt3a signaling by exogenous HPSE in human MB lines possessing increased invasive abilities (D721 > D556 > D283). We identified GEF-H1, a small GTPase inhibitor, as a new component of syndecan-4 - associated signaling complex. Secondly, we demonstrated that HPSE modulated the expression and localization of GEF-H1, β-catenin, and N-Myc in a Shh/Wnt3a - dependent manner, altering MB cell proliferation. Thirdly, we showed that corresponding, GEF-H1 - regulated, small GTPase (Rac/Rho) activities are stimulated differently among MB lines and promote cell invasiveness. Thus, the HSPG/HPSE axis is implicated in two critical MB cell signaling systems and biological outcomes - proliferation and invasion, and potentially relevant as novel target for treatment of invasive MB.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3190.
Abstract 3193: EPHB2 expression parallels malignant behavior in mesothelioma
Authors: Goparaju Chandra M. , Donington Jessica S. , Hirsch Nathalie , Harrington Ryan G. , Pass Harvey I.
Published: February 2011

Purpose: Erythropoietin-producing hepatocellular (EPH) receptors and their Ephrin (EFN) ligands are members of the transmembrane receptor tyrosine kinase (RTK) family. EPHB2 is a RTK, promotes uncontrolled cell growth, migration and tumor progression. Preliminary gene array data indicate EPHB2 overexpression in malignant pleural mesothelioma (MPM). Here, we aim to verify EPHB2 overexpression in MPM tumors and cell lines and hypothesize that EPHB2 silencing decreases malignant behavior in MPM cell lines.

Methods: EPHB2 mRNA expression was evaluated by RT-PCR in eight MPM and three benign mesothelial cell lines, and seven pairs of MPM tumors and matched normal tissue. Functional significance of EPHB2 was evaluated by transfecting EPHB2-shRNA and empty vector controls into two MPM cell lines, HP-1 and H2595. Expression of EPHB2, BCL2L1, survivin, Caspase2, Caspase8, matrix metalloproteinase-2 (MMP-2) and vascular endothelial growth factor (VEGF) were evaluated by RT-PCR following silencing. Pooled populations of transfected cells were evaluated in scratch closure, cell proliferation by CellTitre BlueTM, MatrigelTM Invasion and colorimetric TUNELTM apoptotic assays. Comparisons were made between EPHB2 silenced and control cell lines.

Results: EPHB2 was overexpressed in all MPM cell lines, but not in the benign mesothelial cell lines and in all tumors compared to matched normal tissue. Targeted knockdown of EPHB2 in MPM cell lines reduced its expression and subsequently reduced MMP-2, VEGF, BCL2L1 and survivin expression, and increased Caspase2 and Caspase8 expression. Inhibition of EPHB2 significantly decreased scratch closure, proliferation and invasion compared to the controls, and significantly increased apoptotic cells, likely related to Caspase2 and Caspase8 mediated cell death.

Conclusion: Silencing of EPHB2 reduced the malignant behavior in MPM cell lines. EPHB2 appears to play an important role in MPM pathogenesis and could potentially serve as a novel therapeutic target for MPM treatment.Results of Functional AssaysAssayHP-1 ControlHP-1 EPHB2shRNAp ValueH2595 ControlH2595 EPHB2shRNAp ValueScratch Assay (% closure)69.237.6<0.00556.945.7<0.03Cell Proliferation Assay (OD value)9.56.4<0.058.75.7<0.05Matrigel Invasion Assay (cells invaded)14678<0.0057643<0.005TUNEL Apoptotic Assay (% apoptotic cells)074n/a088.5n/a       

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3193.
Abstract 3207: Telomerase inhibition affects fludarabine sensitivity in quiescent primary chronic lymphocytic leukemia lymphocytes
Authors: Shawi May , Amrein Lilian , Gryaznov Sergei , Autexier Chantal , Aloyz Raquel
Published: February 2011

B- Cell chronic leukemia (CLL) is the most common leukemia in adults. It is clinically very heterogeneous and has a highly variable clinical course. Telomere length and telomerase activity have been shown to be excellent prognostic factors in CLL. Moreover, telomere maintenance is deficient in CLL lymphocytes. These studies have prompted the assessment of the telomerase inhibitor GRN163L in a phase I-II clinical trial in CLL and lymphomas. Here we report that telomerase activity is not required for the survival of quiescent primary CLL lymphocytes. In contrast telomerase activity seems to be required for the survival of quiescent primary CLL lymphocytes after treatment with fludarabine (FLU) in vitro. Furthermore, the effect of fludarabine on telomerase activity was associated with the basal expression of the telomere shelterin protein TRF2. In summary, our results suggest that GRN163L in combination with FLU may be useful to decrease the tumor burden in CLL. To our knowledge this is the first report assessing the effect of telomerase inhibition in combination with a chemotherapeutic agent in non-stimulated primary CLL lymphocytes.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3207.
Abstract 3229: Biomarker validation for a rare malignancy in a mouse model
Authors: Servais Elliot L. , Colovos Christos , Suzuki Kei , Rodriguez Luis , Sima Cami , Rusch Valerie W. , Sadelain Michel , Adusumilli Prasad S.
Published: February 2011

Purpose:

Malignant pleural mesothelioma (MPM) is a rare malignancy often diagnosed at an advanced stage. Clinical validation of a biomarker that would allow for earlier diagnosis as well as detection of recurrence following therapy is limited by the rarity of the tumor. Soluble mesothelin-related peptide (SMRP), which can be detected in patients’ serum, is a cleavage product of mesothelin, a tumor-associated cell surface protein over-expressed in >80% of MPMs. The purpose of this study is to validate the pharmacodynamics of SMRP as a tumor biomarker of prognosis and therapy response using a clinically and pathologically relevant orthotopic xenograft model of MPM.

Experimental Design:

Orthotopic MPM xenograft mice (athymic nude and SCID-beige) were established by direct pleural injection of human mesothelioma cells (MSTO-211H) retrovirally transduced to stably express mesothelin and the GFP-Firefly Luciferase fusion gene. Quantitative bioluminescence imaging (BLI) and MRI were used to serially monitor tumor burden and progression and were correlated with serum SMRP levels (Mesomark® ELISA assay). Kaplan-Meier survival analyses were performed following xenografting of mesothelin and non-mesothelin expressing tumors. The utility of SMRP as a biomarker of therapeutic response was validated by treating MPM mice with either: (a) Cisplatin (4mg/kg IP weekly), (b) thoracic radiation (20Gy total in 5 fractions), (c) combination chemoradiation, or (d) no treatment and correlating serial SMRP levels to survival and BLI signal.

Results:

MPM cells secreting SMRP demonstrated increased invasion and migration in vitro as assessed by the modified Boyden Chamber assay compared to cells without SMRP secretion, 124 vs 48 cells per high-powered field (p<0.0001) and 49 vs 32 cells (p=0.017), respectively. In MPM xenograft mice, increasing SMRP levels correlated with tumor progression by quantitative BLI (spearman r=0.89, p=0.04) and decreased survival (p=0.001). SMRP demonstrated improved sensitivity for detecting early MPM tumors compared to MRI. SMRP levels significantly decreased following treatment versus control mice (p=0.03) and were predictive of survival (hazard ratio = 4.5, 95%CI 1.53-13.1). In addition, SMRP detected tumor recurrence following treatment earlier than MRI.

Conclusions:

We have shown that a serum biomarker of tumor progression and therapy response can be successfully studied in a clinically relevant animal model. To our knowledge, this is the first report utilizing BLI as a reproducible tool, in conjunction with an orthotopic animal model, to quantitatively assess pleural tumor burden and biomarker pharmacodynamics.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3229.
Abstract 3263: Acceleration of mammary tumorigenesis by PDK1 and peroxisome proliferator-activated receptorδ
Authors: Pollock Claire B. , Yin Yuzhi , Yuan Hongyan , Zeng Xiao , King Sruthi , Kopelovich Levy , Glazer Robert I.
Published: February 2011

The 3-phosphoinositide-dependant kinase-1 (PDK1) is a master regulator of least 23 members of the AGC protein kinase family, including protein kinase B (PKB/Akt). Genetic alterations which activate PI3K/AKT signaling are common in cancer, and elevated expression of PDK1 has been detected in various cancer types, including breast cancer. To assess the effect of PDK1 over-expression in mammary gland function and tumorigenesis, transgenic mice were generated that express PDK1 in the mammary epithelium under the control of the MMTV promoter. PDK1 over-expression resulted in increased pT308Akt and pS9GSK3β and PPARδ expression, but did not affect the phosphorylation of other targets, such as RSK2, S6K and BAD. Transgenic mice exhibited a moderate acceleration of tumor induction by progestin/DMBA in comparison to wild-type mice, and GW501516 treatment accelerated tumorigenesis in both transgenic and wild-type animals. GW501516 promoted the formation of highly keratinized squamous cell carcinomas in both PDK1 transgenic and wild-type mice, and exhibited a profile of elevated pT308Akt and PPARδ expression. Treatment of wild-type mice with PPARδ agonist GW501516 alone increased PDK1 and pT308Akt expression in mammary epithelium. Thus, MMTV-PDK1 mice provide a model in which PDK1 enhances mammary carcinogenesis through activation of Akt and PPARδ to elicit a cooperative effect in promoting tumorigenesis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3263.
Abstract 3269: Calpain knockdown in human breast carcinoma cells reduced xenograft tumor growth rates and inhibited in vitro cell migration and proliferation
Authors: Hu Jing H. , Elliott Bruce , Greer Peter A.
Published: February 2011

Ubiquitously expressed µ- and m-calpain are calcium dependent intracellular cysteine proteases that have been implicated in the regulation of cell proliferation, survival, apoptosis, migration, and invasion. Stable expression and proteolytic activity of the µ- and m-calpain catalytic subunits (encoded by capn1 and capn2 genes, respectively) requires a common regulatory subunit encoded by the capn4/capns1 gene; thus both µ- and m-calpain activities can be blocked by genetic knockout or knockdown of capn4 expression. Calpain is also regulated by an endogenously expressed inhibitor peptide, calpastain.

We screened a panel of human breast cancer and normal epithelial cell lines for expression of calpain and calpastatin. µ- and m-calpain and calpastatin were abundantly expressed in MDA-MB-231, SKBR3, T47D, and MCF-7 breast cancer lines, as well as the normal human breast epithelial cell line MCF-10A.

A stably expressed lentivirally encoded shRNA directed against the capn4 transcript was used to effectively knockdown µ- and m-calpain expression and activity in MDA-MB-231 breast cancer cells. This correlated with attenuated proliferation, migration and invasion, but resistance to anoikis. Biochemical analysis also suggests a role for calpain in the regulation of focal adhesion complex proteins such FAK, PTP-1B, talin and F-actin.

Finally, knockdown of calpain in MDA-MB-231 cells significantly reduced their ability to form tumors when orthotopically injected into the mammary fat pads of nude mice. These observations provide direct genetic evidence that calpain plays a pro-tumorigenic role in the MDA-MB-231 breast cancer cell model system.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3269.
Abstract 3271: The RNA binding protein HuR controls angiogenesis in triple negative breast cancer
Authors: Atasoy Ulus , Gubin Matthew , Calaluce Bob , Davis Wade , Magee Joseph , Strouse Connie , Shaw Daniel , Hoffman Timothy , Rold Tammy
Published: February 2011

Due to the poor correlation between steady state mRNA levels and protein products, traditional microarray analysis may miss many genes which are regulated primarily at the level of mRNA stability and translation. Posttranscriptional gene regulation mediated by microRNAs and RNA binding proteins (RBPs) is being recognized as an important form of gene regulation. The elav (embryonic lethal abnormal vision) family of RBPs, are paraneoplastic antigens, over-expressed in a variety of malignancies, including breast cancer. Antibodies against elav family members are believed to be cancer-protective. The elav family binds to the AU-rich elements (AREs) found in the 3’ untranslated regions (UTRs) of many early-response genes, including proto-oncogenes and cell cycle regulators. HuR, the ubiquitously expressed family member, has been described to play a role in cancer progression by stabilizing and translationally up regulating expression of its target mRNAs. Elevated levels of cytoplasmic HuR directly correlate with increased invasiveness of malignancy and poor prognosis for many cancers, including those of the breast. HuR has been described to positively control the expression of multiple genes in the acquired capabilities model, such as VEGF and HIF1α. Hence, it has been suggested that HuR may serve as a tumor maintenance gene which allows for cancers to proliferate. Therefore, it is of interest to discover in vivo HuR targets, as these genes may play vital roles in transformed cells. We have developed methods called RNA immunoprecipitations applied to microarrays, RIP-Chip. We used RIP-Chip to identify distinct subsets of HuR associated mRNAs in MDA-MB-231 and MCF-7 breast cancer cell lines and validated several novel targets. To further investigate the role of HuR in triple negative breast cancer, we over expressed HuR in MDA-MB-231 cells, which results in accelerated growth and alterations in cell cycle kinetics. Surprisingly, when employed in orthotopic mouse models of cancer, HuR over expression significantly inhibited growth of triple negative tumors by 90%. Putative mechanisms appear to be anti-angiogenic, as HuR over expression increases anti-angiogenic factors, but surprisingly, also down regulates pro-angiogenic factors such as VEGF. These results are highly significant because they implicate HuR as a master regulator of angiogenesis in triple negative breast cancer tumor formation and suggest potentially novel treatment methods for this aggressive form of breast cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3271.
Abstract 3290: Overexpression of Aurora B kinase is correlated with vascular invasion and metastasis of human hepatocellular carcinoma
Authors: Lin Zhong-Zhe , Jeng Yung-Ming , Hu Fu-Chang , Pan Hung-Wei , Tsao Hsin-Wei , Hsu Hey-Chi , Cheng Ann-Lii
Published: February 2011

Background:

We previously demonstrated that Aurora B overexpression correlated well with higher histology grade and more advanced stage of hepatocellular carcinoma (HCC). In this study, we sought to analyze the association between Aurora B overexpression and vascular invasion/metastasis of HCC, as well as the potential of AZD1152, a novel and selective Aurora B kinase inhibitor in preventing vascular invasion and metastasis of HCC.

Method:

Aurora B mRNA levels were measured in HCC and paired non-tumorous liver tissues by reverse transcription-PCR. One hundred and sixty surgically resected, primary unifocal HCCs were selected for this study. Vascular invasion was determined by pathologic examination. All 160 patients had been followed for more than 5 years or until death. Forty-nine patients developed extrahepatic tumor metastasis. Multivariate analysis was conducted to determine the significance of Aurora B overexpression on vascular invasion and metastasis of HCC. The effects of AZD1152 on tumor invasiveness were tested in Huh-7 and HA-27T HCC cell lines by migration and invasion assays.

Results:

Overexpression of Aurora B was found in 98 of the 160 (61%) primary HCC. Univariate analysis showed that vascular invasion and metastasis were both associated with younger age (≤ 55 years, P = 0.027 and P = 0.003, respectively), high α-fetoprotein levels (> 200 ng/mL, P < 0.001 and P = 0.008), large tumor size (> 5 cm, P < 0.001 and P < 0.001), higher tumor grade (grade III-IV, P < 0.001 and P = 0.025), as well as Aurora B overexpression (P < 0.001 and P < 0.001). Multivariate analysis confirmed that Aurora B overexpression was an independent risk factor associated with vascular invasion (odds ratio, 2.659; P = 0.0183) and metastasis (odds ratio, 4.195; P = 0.0027). AZD1152, 5-125 nM, induced dose-dependent inhibition on migration and invasion in Huh-7 and HA-27T cells.

Conclusions:

Overexpression of Aurora B correlates well with vascular invasion and metastasis of HCC. Inhibition of Aurora B kinase by small molecular inhibitors may suppress invasion and metastasis of HCC.

Grant support:

Grant NSC96-2628-B-002-054-MY3 from the National Science Council (Taiwan), Grant DOH97-TD-B-111-001 from the Department of Health (Taiwan), and Grant NSC98-3112-B-075A-001 from the National Science Council (Taiwan).

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3290.
Abstract 3308: Differential expression of Notch-1 and Notch-2 correlates to invasive phenotype in bladder cancer cell lines
Authors: Gust Kilian M. , Awrey Shannon , Ettinger Susan , Li Estelle , Fazli Ladan , Black Peter C.
Published: February 2011

The Notch pathway plays a critical role in growth and differentiation, and is involved in cancer progression in different organ sites. Little is known about Notch in bladder cancer. In this study, we evaluated Notch-1 and Notch-2 expression in a panel of bladder cancer cell lines.

Thirteen bladder cancer cell lines with previously described invasive properties and epithelial/mesenchymal characteristics were screened for Notch-1 and Notch-2 expression by Western blot and real-time PCR. Immunohistochemistry (IHC) was performed on a tissue microarray (US Biomax, Inc., Rockville, MD) that included cores from 152 patients with bladder cancer. Alterations in gene expression after Notch-2 silencing with siRNA were measured by expression microarray in 3 cell lines. Genes that changed by at least 2-fold in all 3 cell lines were selected for further analysis. Wound healing assays were performed after treatment with Notch-2 siRNA versus scrambled siRNA. The Mann-Whitney test was performed for statistical analysis.

Epithelial cell lines with low invasive potential expressed Notch-1 at high and Notch-2 at low relative levels (P=0.03 and P<0.01, respectively, compared to invasive cell lines). Highly invasive, mesenchymal cell lines expressed high levels of Notch-2 and low levels of Notch-1. These results were validated by Western blot and IHC (TMA). Microarray analysis revealed altered expression of genes related to epithelial to mesenchymal transition (EMT), protein and endosome trafficking, and cell-cell connections, when Notch-2 was silenced with siRNA. Notch-2 silencing decreased the migration of UC3 cells in the wound healing assay (P=0.02).

These results provide preliminary evidence for a role for Notch in the progression of bladder cancer and it is suggested that this may be related to EMT. Further investigation is needed to assess the suitability of Notch as a therapeutic target, and it would appear that selective inhibition of individual Notch receptors may be preferential over global inhibition.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3308.
Abstract 3332: Engineering functions in cell transduction domains: novel anticancer agents
Authors: Epenetos Agamemnon A. , kousparou christina , Stylianou Spyros , deonarain mahendra
Published: February 2011

Trojantec research has focused on the design of recombinant proteins that overcome both extracellular and intracellular limitations of other delivery systems by rapidly releasing proteins into the cytoplasm and mediating efficient nuclear translocation.

We have genetically engineered two fusion proteins; The first consisting of the Drosophila transcription factor Antennapedia (ANTP) and the truncated version of Mastermind-like (MAML) that behaves in a dominant negative (DN) fashion and inhibits Notch activation (ANTP/DN MAML) (TR4), and the second which is comprised of ANTP fused to the tumour suppressor protein p21, which is important in many key cell division processes including G1-arrest leading to apoptosis of damaged cells.

Our results indicate that drug delivery using with the ANTP cell transduction domain induces apoptosis, abolishes colony formation in soft agar, and inhibits tumor growth in mice, without organ or systemic toxicity. These studies establish TR1 and TR4 as new cancer therapeutic agents in clinical applications.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3332.
Abstract 336: Blockade of CCN6 (WISP3) activates growth factor-independent survival and resistance to anoikis in human mammary epithelial cells
Authors: Huang Wei , Gonzalez Maria E. , Toy Kathy , Banerjee Mousumi , Kleer Celina G.
Published: February 2011

CCN6 is a secreted cysteine rich matricellular protein (36.9 kDa) that exerts growth inhibitory functions in breast cancer. Reduction or loss of CCN6 protein has been reported in invasive carcinomas of the breast with lymph node metastasis and in inflammatory breast cancer. However, the mechanism by which CCN6 loss promotes breast cancer growth remains to be defined. In the present study we developed lentiviral mediated shRNA knockdown of CCN6 in nontumorigenic mammary epithelial cells MCF10A and HME. We discovered that CCN6 knockdown protects mammary epithelial cells from apoptosis and activates growth factor-independent survival. In the absence of exogenous growth factors, CCN6 knockdown was able to promote growth under anchorage independent conditions and triggered resistance to detachment-induced cell death (anoikis). Upon serum starvation, CCN6 knockdown was sufficient for activation of the phosphatidylinositol 3-kinase/Akt pathway. Growth factor-independent cell survival was stunted in CCN6 knockdown cells when treated with either human recombinant CCN6 protein or the phosphatidylinositol 3-kinase inhibitor LY294002. Targeted inhibition of Akt isoforms revealed that the survival advantage rendered by CCN6 knockdown requires specific activation of Akt-1. The relevance of our studies to human breast cancer is highlighted by the finding that low CCN6 protein levels are associated with upregulated expression of phospho-Akt-1 (Ser473) expression in 21% of invasive breast carcinomas. These results enable us to pinpoint one mechanism by which CCN6 controls survival of breast cells mediated by the PI3K/Akt-1 pathway.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 336.
Abstract 338: Enhanced cetuximab and radiosensitization with src inhibition in head and neck cancer
Authors: Li Chunrong , Iida Mari , Dunn Emily , Wheeler Deric L.
Published: February 2011

The epidermal growth factor receptor (EGFR) is a ubiquitously expressed receptor tyrosine kinase (RTK) that initiates a spectrum of signaling pathways that promote cell proliferation, differentiation, migration, motility, and metastasis. The EGFR has been strongly linked to the etiology of head and neck squamous cell carcinoma (HNSCC). The first major phase III clinical trial combining cetuximab with radiation in HNSCC confirmed a strong survival advantage for patients on the cetuximab arm. It has recently been reported, however, that cetuximab and radiation can induce EGFR to the nucleus where it can enhance resistance to both of these therapeutic modalities. In this report we sought to determine how to block cetuximab and radiation induced translocation of EGFR to the nucleus to increase the efficacy of this therapeutic regimen in HNSCC.

We utilized three established HNSCC lines, SCC1, SCC6 and SCC1483 and measured nuclear translocation of the EGFR after treatment with cetuximab, radiation or the combination. Both cetuximab and radiation treatment could induce nuclear translocation of the EGFR in all SCC lines tested. Strikingly, time course analysis indicated that cetuximab could stimulate movement of the EGFR to the nucleus within one hour where it maintained its present for greater than 96 hours. Conversely, radiation could stimulate EGFR translocation to the nucleus within 0.5 hours, but returned to baseline levels within four hours. Both cetuximab and radiation treatment of SCC lines led to the phosphorylation of tyrosine 845 (Y845) of the EGFR. Y845 is phosphorylated specifically by the non-receptor tyrosine kinase Src and/or its family of kinases (SFKs). Blockade of SFKs by dasatinib could abrogate cetuximab and radiation induced EGFR translocation to the nucleus indicating that SFKs are crucial for the movement of the EGFR to the nucleus. In addition, pre-treatment of HNSCC cells with dasatinib, followed by cetuximab and radiation enhanced the suppression of ERK activity in all three SCC cell lines. Furthermore, combining dasatinib with cetuximab/radiation treatment exhibited additive inhibitory effects on cell growth or clonogenic formation. These data suggest that both cetuximab and radiation can induce EGFR to the nucleus and blockade of SFKs using dasatinib can abrogate this translocation. Collectively these findings may suggest that dasatinib can limit EGFR translocation to the nucleus and may enhance radiotherapy plus cetuximab in HNSCC.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 338.
Abstract 3390: Cas-L promotes migration, invasion and recurrence in human lung cancer
Authors: Kondo Shunsuke , Iwata Satoshi , Inoue Yusuke , Yamada Taketo , Morimoto Chikao
Published: February 2011

Lung cancer is the most common cause of cancer mortality world wide. Surgical intervention is currently the most effective treatment modality for non-small cell lung cancer (NSCLC) confined to the thorax. The relapse rate among patients with early-stage NSCLC is 40% within 5 years after potentially curative treatment. Adjuvant chemotherapy is added to standard therapy, but the efficacy is limited. To identify molecular subtypes associated with outcome facilitates selecting patients who can be offered an additional treatment option

Crk-associated substrate lymphocyte type (Cas-L) is a docking protein that is heavily tyrosine phosphorylated via β1 integrins and growth factor dependent signals in cancer cells and lymphocytes. Cas-L plays a role in biologically regulatied cell attachment, migration, invasion, apoptosis and cell cycle and also plays a role in the development of metastatic capability in cancer. Cas-L protein is identified with an essential switch for pro-metastatic behavior in tumors. Cas-L mRNA and protein expression were elevated in a significant percentage of metastatic melanomas and that this elevated Cas-L expression was required for the metastatic process. Here in, we showed the potential role of Cas-L in the phosphorylation and downstream signaling of epidermal growth factor (EGF) receptors in NSCLC. Cas-L is involved in EGF-stimulated migration and invasion of NSCLC cells. Additionally, we suggest that phosphorylation of Cas-L and the SH3 domain of Cas-L, and binding with docking proteins such as Src kinase and FAK, are necessary for EGF-stimulated migration and invasion.

An in vivo mouse model showed Cas-L over-expression to promote tumorgenesis and metastasis. In clinical samples, our results show that Cas-L promotes invasion in N2 lymph nodes (p≡0.02), pathological lymphatic invasion (p≡0.01) and pathological venous invasion (p≡0.001). Furthermore, Cas-L is associated with recurrence (Hazard ratio 4.24, 95%CI 1.93-9.26; p<0.001) and prognosis (Hazard ratio 5.35, 95%CI 1.98-14.50, p≡0.001).

In conclusion, Cas-L may be useful in selecting patients for adjuvant chemotherapy and serve as an important therapeutic target in NSCLC, as a recurrence marker.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3390.