bluuebunny/crossref_metadata_2025 huggingface dataset:
Humic Acid Removal from Aqueous Environments by Electrocoagulation Process Using Iron Electrodes
Authors: Bazrafshan Edris , Biglari Hamed , Mahvi Amir Hossein
Published: May 2012

At present study the performance of electrocoagulation process using iron electrodes sacrificial anode has been investigated for removal of HA from artificial aqueous solution. The experiments were performed in a bipolar batch reactor with four iron electrode connected in parallel. Several working parameters, such as initial pH (3, 5, 7, and 9), electrical conductivity (50 V) and reaction time were studied in an attempt to achieve the highest removal capacity. Solutions of HA with concentration equal 20 mg L-1 were prepared. To follow the progress of the treatment, samples of 10 ml were taken at 15, 30, 45, 60, and 75 min interval. Finally HA concentration was measured by UV absorbance at 254 nm (UV254) and TOC concentration was measured by TOC Analyser. The maximum efficiency of HA removal which was obtained in voltage of 50 V, reaction time of 75 min, initial concentration 20 mg L-1, conductivity 3000 µS/Cm and pH 5, is equal to 92.69%. But for natural water samples at the same optimum condition removal efficiency was low (68.8 %). It can be concluded that the electrocoagulation process has the potential to be utilized for cost‐effective removal of HA from aqueous environments.
Effect of Magnetite Nano Particles on p-n‐Alkyl Benzoic Acid Mesogens
Authors: Sastry S. Sreehari , Begum S. Salma , Kumari T. Vindhya , Murthy V. R. K. , Ha Sie Tiong
Published: May 2012

The magnetite (Fe3O4) nanoparticles (0.5 wt%) of size less than 20 nm doped in p-n‐alkyl benzoic acids where n varies from heptyl (7) to nonyl (9) are prepared and the presence of Fe+3 is confirmed through UV‐Visible spectrophotometer. Textural and phase transition temperature studies are carried out using polarizing optical microscopy on pure and nano doped p-n‐alkyl benzoic acids. These results are further confirmed by DSC at a scan rate of 5ºC/min and dielectric studies. Dielectric studies are carried out, in which the variation of dielectric constant, loss and the conductivity are analyzed with respect to temperatures and frequencies. Increment of relaxation times for nano doped heptyl and nonyl benzoic acids are observed which implies that the dielectric nature is strengthened for the nano doped mesogens. The preference of nano doped p-n‐alkyl benzoic acids is discussed.
Synthesis, Spectral and Thermal Behavior of Some New Four Coordinated Complexes
Authors: Montazerozohori M. , Khani S. , Joohari S. , Musavi S. A.
Published: May 2012

A new symmetric bidentate Schiff base N, N′ bis(4‐fluorobenzal‐dehydene)‐1,2‐diaminoethane(L) and its complexes with general formula MLX2 (M= Zn(II), Cd(II) and Hg(II) and X=chloride, bromide, iodide, thiocyanate and azide) have been prepared. The ligand and complexes have been established by microanalysis, electronic, FT‐IR, 1H and 13C NMR spectra, and by molar conductivity measurements. All compounds are non‐electrolytes in chloroform or DMSO‐d6. The thermal behavior of the complexes shows weight loss by decomposition of the anions and ligand segments in the subsequent steps. Some activation thermodynamic parameters such as E*, ∆H*, ∆S* and ∆G* were calculated from thermal analysis.
Abstract A10: Increased mesenchymal progenitor cells in RB1−/− bone
Authors: Kong Elizabeth , Gutierrez Gabriel , Thomas David , Hinds Philip
Published: May 2012

The retinoblastoma pathway is deregulated in 90% of human cancers owing to the mutation of one or more pathway members. Yet, the molecular pathogenesis of tumors with a loss of the retinoblastoma gene, RB1, (retinoblastoma and osteosarcoma) are poorly understood. To understand the role of pRb in tumor formation we have focused on a tissue in which malignancies commonly have RB1 deletions, bone. Human osteosarcoma cells, in general, exhibit no signs of terminal differentiation, and are defined by the ability to form bone. We hypothesize that osteosarcoma is caused by molecular/genetic disruptions of the osteogenic differentiation pathway. Homozygous null RB1 mice die during early embryonic day, E11.5-13.5, before any significant skeletal development or mineralization occurs. Therefore, we utilized the cre/lox system to create a bone targeted RB1 knockout animal. RB1−/− mice display defects in bone formation. Furthermore, isolation and characterization of primary calvarial cells (dorsal skull cells) from these animals show that pRb abates progression of pre-osteoblasts to mature osteoblasts. As osteoblasts are known progeny of primitive mesenchymal stem cells (MSC), we next asked if diminished differentiation resulted in an increase in MSCs. MSCs are pluripotent cells that can differentiate into a variety of cell types. I have shown in the absence of RB1, calvarial cells can differentiate into multiple lineages including bone and fat, where as cells fromWT littermates are restricted to the osteoblast lineage. Colony formation assays, a functional in vitro assay, suggest RB1−/− cells have increased progenitor cell number, and through sphere formation assays we have shown an increased self-renewal capacity. There is also a significantly larger population of Sca-1 (a stem cell marker) positive cells in RB1−/− cultures compared toWT littermates. In addition, utilization of microarray technology suggests a unique RB1 dependent gene expression pattern in osteogenesis. This is consistent with our previous work showing the necessity of the pRb/Runx2 (a master regulator in bone development) interaction to turn on osteocalcin transcription, a late marker in differentiation. We have begun to identify a possible mechanism for pRb regulation early in osteogenesis through the Hedgehog signaling pathway that explains the increased pluripotency of mesenchymal cells in the absence of RB1. Therefore, we have identified an increased number of bone progenitor cells that are pluripotent from RB1−/− mice, and a possible mechanism for pRb regulation early in osteogenesis and malignant tumor formation.

Citation Information: Cancer Res 2009;69(23 Suppl):A10.
Abstract A12: YAP-dependent induction of amphiregulin identifies a non-cell-autonomous component of the Hippo pathway
Authors: Zhang Jianmin , Ji Jun-Yuan , Yu Min , Overholtzer Michael , Smolen Gromoslaw A. , Brugge Joan S. , Dyson Nicholas J. , Haber Daniel A.
Published: May 2012

The Hippo signaling pathway was initially identified in Drosophila and components in this pathway are highly conserved in mammals. We and others have recently shown that the Hippo pathway regulates cellular proliferation and survival, thus exerting profound effects on normal cell fate and tumorigenesis. The pivotal effector of this pathway is YAP, a transcriptional co-activator amplified in mouse and human cancers, where it promotes epithelial to mesenchymal transition (EMT) and malignant transformation. To date, studies of YAP target genes have focused on cellautonomous mediators. Here, we show that YAP-expressing MCF10A breast epithelial cells secrete growth factors that enhance the proliferation of neighboring untransfected cells, implicating a non-cell autonomous mechanism. Using cytokine and growth factor array analysis, we identified the epidermal growth factor receptor (EGFR) ligand, amphiregulin (AREG), as a transcriptional target of YAP, whose induction contributes to YAP-mediated cell proliferation and migration. Knockdown of AREG or addition of an EGFR kinase inhibitor abrogates the proliferative effects of YAP expression. Suppression of the negative YAP regulators LATS1/2 is sufficient to induce AREG expression, consistent with a physiological regulation of AREG by the Hippo pathway. Furthermore, genetic interactions between the Drosophila YAP orthologue Yki and Egfr signaling components support the link between these two highly conserved signaling pathways. In conclusion, YAP-dependent secretion of AREG implicates activation of EGFR signaling as an important non-cell autonomous effector of the Hippo pathway, with relevance for the regulation of both physiological and malignant cell proliferation.

Citation Information: Cancer Res 2009;69(23 Suppl):A12.
Abstract A19: Enhanced leptin receptor expression identifies an aggressive subset of Middle Eastern papillary thyroid cancer
Authors: Al-Kuraya Khawla S. , Bavi Prashant , Siraj Abdul K. , Uddin Shahab
Published: May 2012

The putative role of leptin (Ob) and its receptor (Ob-R) in the pathogenesis of various primary human malignancies has been investigated in recent studies. However, the role of leptin and leptin receptor (Ob-R) in papillary thyroid cancer (PTC) has not yet been evaluated. In this study we investigated role of Ob-R in a large tissue microarray cohort of PTC followed by in vitro studies using a panel of PTC cell lines. Ob-R overexpression was seen in 80% PTCs and was significantly associated with poor disease free survival (p=0.0235). PTCs that overexpressed Ob-R showed a aggressive phenotype characterized by older age, extrathyroid extension, larger tumor size, nodal metastasis, advanced stage and tall cell variant histological subtype (p=0.0005, p=0.0006, p=0.0398, p= 0.0004, p=0.0111 and p=0.0003 respectively). Further PTCs with overexpression of Ob-R showed a significant direct association with overexpression of leptin (p=0.0004), XIAP (p<0.0001) and BCL-XL (p<0.0001). In vitro analysis showed that leptin stimulated cell proliferation and inhibited apoptosis via activation of phosphatidylinisitol 3-kinase (PI3K)/AKT signalling pathway. Inhibition of PI3K activity by LY294002, a specific inhibitor of PI3K kinase abrogated leptin-mediated PI3K/AKT signalling. Gene silencing of Ob-R with Ob-R siRNA in PTC cells resulted in down regulation of phospho-AKT, Bcl-XL and XIAP expression suggesting that leptin mediated pathogenesis of PTC occur via involvement of these downstream targets. Altogether these data suggest that leptin plays a critical role in PTC pathogenesis through PI3K/AKT pathway via Ob-R and is a potential prognostic marker associated with an aggressive phenotype and poor disease free survival.

Citation Information: Cancer Res 2009;69(23 Suppl):A19.
Abstract A20: Protein kinase C beta II is crucial for Ewing sarcoma cell survival
Authors: Surdez Dider , Benetkiewicz Magdalena , Perrin Virginie , Delattre Olivier , Tirode Franck
Published: May 2012

Ewing sarcoma, the second most frequent bone tumor of young adults, is most frequently characterized by a specific translocation t(11;22) leading to the formation of the aberrant transcription factor EWS-FLI1. By analyzing transcriptional profiles of Ewing tumors with other pediatric tumors, we identified genes that were highly and very specifically over-expressed in Ewing sarcomas, as compared to the others. Beside already identified EWS-FLI1 targets like NKX2-2 or NR0B1, we focused our attention on a novel potential EWS-FLI1 target gene: the protein kinase C beta (PRKCB). Firstly, we confirmed at the protein level, that PRKCB is highly expressed in different Ewing cell lines. Secondly, EWS-FLI1 shRNA inhibition experiments in different Ewing cell lines demonstrated that the PRKCB over-expression is dependent on the expression of the fusion protein. Thirdly, using luciferase reporter experiments together with chromatin immunoprecipitation, we could show that EWS-FLI1 can bind directly to the PRKCB promoter and regulate its activity, demonstrating that PRKCB is a new direct target of EWS-FLI1. Finally, we investigated the role of the two different PRKCB isoforms in Ewing cells by inhibiting either both or the second one only by knock-down experiments. Both strategies led to a dramatic decrease of cell proliferation together with a massive cell death, indicating that at least PRKCB isoform 2, which seems to be the most expressed isoform in Ewing cells, is crucial for Ewing cell survival. We obtained the same results while using specific inhibitor compounds of PRKCB. Altogether, these results show that protein kinase C beta is highly expressed in Ewing tumors and that its inhibition could be of major interest for developing new therapeutic strategies.

Citation Information: Cancer Res 2009;69(23 Suppl):A20.
Abstract A21: MAGE cancer-testis antigens facilitate the degradation of p53 by E3 RING ubiquitin ligases
Authors: Potts Patrick Ryan
Published: May 2012

Cancer testis antigens are aberrantly expressed in cancer cells but absent from normal somatic cells, and as such they constitute excellent targets for cancer immunotherapy. Surprising recent evidence suggests that their aberrant expression in cancer cells is not simply an inert consequence of widespread genomic deregulation, but rather an important functional event promoting the cancer phenotype. However, the mechanism through which these proteins promote cancer remains completely unknown due to the lack of structural or functional characterization. Here we report the structural and functional identity of the MAGE proteins, a large family of cancer testis antigens and ubiquitous proteins of previously unknown function. Through structural prediction, X-ray crystallography, and biochemistry we show that a conserved feature of the MAGE family of proteins is their ability to bind to E3 RING ubiquitin ligases, such TRIM28/KAP1. Surprisingly, MAGE-RING ubiquitin ligases share structural and biochemical similarities to Cullin-RING ubiquitin ligases. Most notably, MAGEs activate the ubiquitin ligase activity of E3 RING ubiquitin ligases. Furthermore, we show that the tumor suppressor p53 is a critical and direct target of MAGE-mediated ubiquitination and degradation in breast cancer. Thus we have identified a novel, cancer-specific regulator of p53 degradation and breast cancer survival and discovered the function of the enigmatic MAGE family of cancer testis antigens. These results underscore the importance of MAGE proteins as therapeutic targets for cancer.

Citation Information: Cancer Res 2009;69(23 Suppl):A21.
Abstract A22: Pooled analysis of phosphatidylinositol 3-kinase (PI3K) pathway variants and risk of prostate cancer
Authors: Koutros Stella , Schumacher Fredrick R. , Ma Jing , Huang Wen-Yi , Hayes Richard B. , Berndt Sonja I.
Published: May 2012

Background: The phosphatidylinositol 3-kinase (PI3K) pathway regulates various cellular processes such as cell growth, proliferation, apoptosis, motility, differentiation, survival and intracellular trafficking. In addition, numerous growth factors signal through the PI3K pathway, including insulin-like growth factors, which may impact prostate carcinogenesis. Nevertheless, single nucleotide polymorphisms (SNPs) in PI3K genes have not been extensively studied as risk factors for this disease.

Methods: Pooled data from seven prospective studies in the National Cancer Institute Breast and Prostate Cancer Cohort Consortium were examined for associations between 89 SNPs in five PI3K pathway genes (PIK3C2B, PIK3AP1, PIK3C2A, PIK3CD, and PIK3R3) and prostate cancer risk in 8,309 cases and 9,286 controls. Odds ratios (OR) and 95% confidence intervals (CI) adjusted for age, subcohort, and ethnicity were estimated using unconditional logistic regression.

Results: Several SNPs clustered upstream and in the first two introns of PIK3C2B were related to prostate cancer risk. SNP rs7556371 in PIK3C2B was significantly associated with prostate cancer risk (ORper allele = 1.08 (95% CI: 1.03, 1.14), p-trend = 0.0017) even after adjustment for multiple testing (Padj=0.024). Simultaneous adjustment of rs7556371 with nearby SNPs strengthened the association (ORper allele = 1.21 (95% CI: 1.09, 1.34); p-trend =0.0003). The adjusted association was stronger for men diagnosed < 65 years (ORper allele = 1.47 (95% CI: 1.20, 1.79), p-trend = 0.0001) and for men with a family history (ORper allele = 1.57 (95% CI: 1.11, 2.23), p-trend = 0.0114) but was strongest among men with younger age at diagnosis who also had a family history (ORper allele = 2.31 (95% CI: 1.07, 5.07), p-interaction = 0.005). No differences were observed with disease aggressiveness (Gleason ≥8/stage 3/4/fatal).

Conclusions: We observed an association between PIK3C2B SNPs and prostate cancer risk. Additional adjustment for nearby SNPs showed a stronger association, suggesting other genetic variants in the region may contribute to risk. This region was statistically significantly associated with familial, early onset disease potentially attributable to IGF-dependent PI3K signaling.

Citation Information: Cancer Res 2009;69(23 Suppl):A22.
Abstract A25: Array-based comparative genomic hybridization of hepatocellular carcinoma reveals a unique genomic aberration pattern in tumors with poor prognosis
Authors: Roessler Stephanie , Long Lori , Budhu Anuradha , Chen Yidong , Zhao Xuelian , Walker Robert , Jia Hu-Liang , Ye Qing-Hai , Qin Lun-Xiu , Tang Zhao-You , Hunter Kent , Thorgeirsson Snorri , Meltzer Paul , Wang Xin
Published: May 2012

Hepatocellular carcinoma (HCC) is the third leading cause of cancer death worldwide. Like most solid tumors, HCC is very heterogenous in its clinical presentation and genetic makeup, due to genomic instability. The poor survival of HCC patients is partly due to inefficient treatment regimens and the lack of accurate prognostic subgroup stratifications. The discovery of genomic changes in other cancer types has led to the development of novel specific therapies, underscoring the importance of understanding molecular mechanism and tumor biology to provide the best therapy for each cancer patient. To this end, we performed high-resolution array-based comparative genomic hybridization of 76 HCC clinical specimens (cohort1) to search for genetically disrupted genes associated with outcome. In agreement with previous publications, recurrent gains and losses were associated with 1q, 6p, 8q and 4q, 8p, 13q, 16, 17p, respectively. However, only deletions of a few distinct loci on 1p,4q, 8p and 9p were associated with survival (FDR<0.05). Of the 419 genes which mapped to regions of loss, analysis of cohort1 revealed that gene expression and DNA copy number of 134 genes (31.98%) were significantly correlated (Pearson coefficient r>0.3, p<0.001). Next, we built a 10-gene-survival signature based on the expression of these 134 genes utilizing cohort1 as a training and testing set with validation in an independent cohort2 (N=180). Kaplan-Meier survival analysis revealed that the 10-gene signature was able to predict survival outcome in tumor tissue (log-rank p=0.008) but not in paired non-tumor samples of cohort2 (log-rank p=0.421). Cox proportional hazards regression analysis controlling for potentially confounding clinical covariates showed that the 10-gene signature was significantly associated with a 2.1-fold increased risk of death. In addition, the 10-gene signature was able to predict survival in three publicly available breast cancer cohorts. In this study, we found a 10-gene signature that allows for the stratification of patients with poor prognosis in HCC and breast cancer. Ongoing functional analysis of the genes in this signature may lead to the discovery of HCC subtype-specific drugs that might be used for personalized therapy.

Citation Information: Cancer Res 2009;69(23 Suppl):A25.
Abstract A28: Aberrant c-Myc protein stabilization and Axin1 deregulation in acute leukemia
Authors: Tibbitts Deanne C. , Fleming William , Fan Guang , Sears Rosalie
Published: May 2012

The c-Myc oncoprotein is an important regulator of cellular growth, proliferation, differentiation, and apoptosis, and c-Myc expression is commonly increased in cancer. Historically, increased c-Myc expression in hematopoietic cancers has been explained by gene amplification or chromosomal translocations that deregulate its transcription. Recent work from our lab suggests that deregulated phosphorylation of c-Myc associated with disrupted c-Myc degradation constitutes an additional mechanism by which c-Myc can drive cancer. We have previously shown that c-Myc degradation is regulated by hierarchical phosphorylation at 2 conserved sites, serine 62 (S62) and threonine 58 (T58), and is coordinated by the scaffold protein and tumor suppressor Axin1. Building on our previous work showing that in a variety of human leukemia cell lines c-Myc is aberrantly stabilized with altered phosphorylation at T58 and S62, we are examining the prevalence and potential mechanism of increased c-Myc stability in primary acute myelogenous leukemia (AML), T cell acute lymphoblastic leukemia (ALL), and B cell ALL patient samples. Based on our recent work showing that c-Myc protein stability is negatively regulated by Axin1, we are examining mechanisms of Axin1 deregulation in AML and ALL patient samples. Finally, by manipulating Axin1 in both leukemic and non-transformed hematopoietic cell lines, we will investigate Axin1's role in directly controlling c-Myc stability and phosphorylation in hematopoietic lineages and whether Axin1 may be a potential therapeutic target in acute leukemias.

Citation Information: Cancer Res 2009;69(23 Suppl):A28.
Abstract A3: Facilitating epithelial to mesenchymal transition through inhibition of senescence checkpoint functions
Authors: Ohashi Shinya , Natsuizaka Mitsuteru , Wong Gabrielle , Grugan Katharine D. , Kalabis Jiri , Rustgi Anil K. , Nakagawa Hiroshi
Published: May 2012

Transforming growth factor (TGF)-β is a potent inducer of epithelial to mesenchymal transition (EMT). However, EMT is not a sole consequence of TGF-β mediated stimulation. It remains unclear as to what determines the underlying cellular capacity to undergo EMT in response to TGF-β. Overexpression of epidermal growth factor receptor (EGFR) and mutations in tumor suppressor p53 are common in the early stage of esophageal carcinogenesis and required for transformation and invasive growth of human esophageal epithelial cells. EGFR overexpression and mutant p53 appear to be key determinants in facilitating TGF-β-induced EMT. EGFR tyrosine kinase activity was dispensable for EMT. However, EGFR overexpression fated the esophageal cells to EMT through induction of ZEB1 and ZEB2, zinc finger E-box binding proteins which evade EGFR-mediated oncogenic as well as the TGF-β-activated senescence responses by inhibiting p15INK4b and p16INK4a. Mutant p53 limits TGF-β-induced senescence by suppressing the p53-dependent senescence checkpoint function. When senescence was negated, TGF-β prompted EMT through induction of ZEB1 and ZEB2 along with concomitant downregulation of the microRNA-200 family. Thus, genetic lesions may involve not only suppression of senescence checkpoint functions but also reactivation of the cellular EMT program in the early stages of carcinogenesis.

Citation Information: Cancer Res 2009;69(23 Suppl):A3.
Abstract A32: Combined inhibition of phosphoinositide-3-kinase and mitogen-activated protein kinase pathways prevents activation of feedback loops in cancer
Authors: Sos Martin L. , Fischer Stefanie , Ulrich Roland , Peifer Martin , Thomas Roman K.
Published: May 2012

In tumor cells, genetically activated oncogenes often induce dependency on the constitutive activity of the mutant oncoprotein. Therapeutic inhibitors exploit this dependency by abrogating the oncogenic signaling, inducing massive apoptosis and thus leading to tumor regressions in patients. The phosphoinositide-3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signaling pathways are key regulators of tumor maintenance and oncogenic transformation.

A major hurdle in the transition from preclinical drug discovery to clinical trials lies in the genomic diversity of human tumors and the lack of preclinical models that capture this diversity. Given the impact of genomic aberrations on therapeutic response, such models are necessary in order to identify genetic lesions linked with individual drug response. In order to overcome these limitations we have recently established a panel of 84 NSCLC cell lines that we have characterized in depth in gene copy number, gene expression and mutation space (Sos et al.; J Clin Invest 2009).

In this study, we interrogated whether upstream dependency encodes for a certain “downstream” dependency either in the PI3K- or the MAPK- pathway and analyzed whether such a downstream pathway dependency can be linked to certain genetic lesions. We demonstrate by systematically linking compound activity to genetic lesions in vitro and in vivo, that tumors with genetically activated receptor tyrosine kinases predominantly depend on PI3K signaling while tumors with mutations in the Ras/Raf pathway depend on MAPK signaling. However, release of negative feedback loops limited the efficacy of downstream pathway inhibition. By contrast, combined blockade of both PI3K- and MAPK-pathways was able to block the reciprocal pathway activation induced by inhibitor-mediated release of negative feedback loops and resulted in a significant increase in apoptosis. Using a systematic chemo-genomics approach, we provide a framework for PI3K and MAPK pathway activation in connection with particular genetic aberrations and provide a rationale for combined inhibition of both pathways. Our findings may impact patient stratification in clinical trials involving PI3K and MAPK inhibitors either as single agents or in combination.

Citation Information: Cancer Res 2009;69(23 Suppl):A32.
Abstract A36: Fibronectin promotes glioma progression by modulating survivin expression and regulatory T cell infiltration
Authors: Sengupta Sadhak , Nandi Suvobroto , Rolle Cleo E. , Han Yu , Lesniak Maciej S.
Published: May 2012

Glioblastoma multiforme (GBM) is the most common and lethal primary brain tumor. Current therapies include surgery, radiation and chemotherapy which only modestly improve patient survival. Glioma progression depends upon exponential growth of the tumor cells accompanied by an increase in acute immunosuppression due to tumor infiltration by regulatory T cells (CD4+CD25+FoxP3+, Tregs). Although several markers responsible for glioma progression have been described, none have been identified as key elements responsible for both glioma growth and immunosuppression. In this study we characterize the role of fibronectin, a high-molecular weight extracellular matrix glycoprotein secreted by tumor cells, in controlling glioma progression and mediating immunosuppression. Fibronectin binds to membrane-spanning receptor proteins called integrins and plays an important role in cell signaling and defining cellular shape, mobility, and regulate the cell cycle. Inhibition of fibronectin expression in glioma cells using RNA-silencing delayed cell division in vitro. Analysis of the mechanism for this delayed cell division showed reduced phosphorylation of Src kinase and STAT-3 (Tyr705) resulting in inhibition of survivin, an important marker for glioma progression. In an animal model of glioma, the mean survival of experimental animals increased from 30 days for wild-type tumors to 53 days in fibronectin knockdown tumors. In vitro expression of Treg-recruiting chemokines was reduced in fibronectin knockdown tumor cells. Moreover, brain samples of animals bearing fibronectin knockdown tumors showed delayed infiltration of Tregs. Collectively, we propose that fibronectin is a key marker for glioma progression, as it enhances both tumor progression and immunosuppression.

Citation Information: Cancer Res 2009;69(23 Suppl):A36.
Abstract A44: Src-induced human cell transformation
Authors: Sukezane Taiko , Hanafusa Hidesaburo , Akagi Tsuyoshi
Published: May 2012

Src is one of the oldest and most investigated oncogenes, and its family members make up the largest group of non-recepter tyrosine kinases. The elevated Src expression and kinase activity have been seen in multiple solid tumors including colon and breast cancer, and many in vitro experiments have shown a critical role of Src in cellular growth and proliferation, angiogenesis, and invasion and metastasis. However, there is no obvious mutation of Src has been linked with the development of human cancer unlike activating mutations of Ras were often found in various types of human cancer, and therefore direct involvement of Src in human cancer pathogenesis has still remained obscure. In order to directly assess the oncogenic potential of Src in human cells, we have expressed activated Src (v-Src) in human diploid fibroblasts (HDF) which were immortalized by the combination of human telomerase reverse transcriptase (hTERT) and SV40 early region (ER). These v-src-expressing HDF exhibit more drastic morphological changes, aggressive tumorigenecity in nude mice, and higher activity of both MAPK and PI3K signaling pathways when compared to the equivalent HDF transformed by H-ras V12. Curiously, there is no such difference between v-Src and H-ras V12 in the transformation of rodent fibroblasts. Such highly tumorigenic potential of v-Src is not limited to fibroblasts, and we have succeeded to transform normal human mammary epithelial cells (HMEC) into aggressive tumor cells by the similar procedure as in HDF. These v-Src transformed HMEC should provide a useful model cell system for understanding the molecular basis of some types of human breast cancer.

Citation Information: Cancer Res 2009;69(23 Suppl):A44.
Abstract A45: The prolyl isomerase Pin1 interacts with a ribosomal protein S6 kinase to enhance insulin-induced AP-1 activity and cellular transformation
Authors: Choi Hong Seok , Namgoog Gwang Mo , Cho Hae-Gook
Published: May 2012

Phosphorylation of proteins on serine or threonine residues that immediately precede proline (pSer/Thr-Pro) is specifically catalyzed by the peptidyl-prolyl cis/trans isomerase Pin1 and is a central signaling mechanism in cell proliferation and transformation. Although Pin1 is frequently overexpressed in hepatocellular carcinoma (HCC), the molecular mechanism of Pin1 in HCC has not been completely elucidated. Here, we show that Pin1 interacts with p70S6K in vitro and ex vivo. Overexpression of Pin1 resulted in enhanced p70S6K phosphorylation induced by insulin in SK-HEP-1 cells. In contrast, Pin1−/− mouse embryonic fibroblasts (MEFs) exhibited significantly decreased insulin-induced p70S6K phosphorylation compared to Pin1+/+ MEFs. Furthermore, Pin1 enhanced the insulin-induced ERK1/2 phosphorylation through its interaction with p70S6K, whereas the inhibition of p70S6K activity by rapamycin suppressed insulin-induced ERK1/2 phosphorylation in SK-HEP-1 cells. Hence, Pin1 affected AP-1 activity through p70S6K-ERK1/2 signaling in SK-HEP-1 cells. Most importantly, Pin1-overexpressing JB6 Cl41 cells enhanced neoplastic cell transformation promoted by insulin much more than GFP-overexpressing JB6 Cl41 control cells. These results imply that Pin1 amplifies insulin signaling in hepatocarcinoma cells through its interaction with p70S6K, suggesting that Pin1 plays an important role in insulin-induced tumorigenesis and is a potential therapeutic target in hepatocarcinoma.

Citation Information: Cancer Res 2009;69(23 Suppl):A45.
Abstract A46: Tpl-2 kinase downregulates the activity of p53 and enhances signaling pathways leading to activation of activator protein 1 induced by EGF
Authors: Cho Hae-Gook , Namgoog Gwang Mo , Choi Hong Seok
Published: May 2012

Tumor progression locus-2 (Tpl-2) kinase is a member of the mitogen-activated protein kinase (MAPK) kinase kinase family that has been implicated in cellular transformation. The enhanced expression of this protein has been shown to activate both the MAPK and c-Jun N-terminal kinase (JNK) pathways. However the molecular mechanisms responsible for the oncogenic potential of Tpl-2 are still largely unknown. Here, we show that Tpl-2 interacts with p53 both in vitro and ex vivo. The overexpression of Tpl-2 inhibits the phosphorylation of p53 at serine 15 induced by EGF by upregulating the activity of protein phosphatase 2A (PP2A). In addition, the EGF-induced p53 activity was suppressed in the Tpl-2-wild-type-transfeted HEK 293 cells, but had no effect in the Tpl-2-mutant (S413A)-transfected cells. Furthermore, the Tpl-2 wild type, but not Tpl-2 mutant (S413A), showed increased EGF-induced c-fos promoter activity, followed by AP-1 transactivation activity, which is associated with the cell transformation prompted by the H-Ras-Tpl-2-AP-1 signaling axis. These results suggest that Tpl-2 plays an important role in EGF-induced carcinogenesis by inactivating the p53 tumor suppressor.

Citation Information: Cancer Res 2009;69(23 Suppl):A46.
Abstract A5: Transient TGFβ exposure reprograms mouse mammary epithelial cells in vitro and in vivo
Authors: Seo Jae-Hong , Dunphy Karen A. , Kim Daniel , Roberts Amy L. , Yan Haoheng , Tao Luwei , Hagen Mary J. , Jerry D. Joseph
Published: May 2012

Transforming growth factor beta (TGFβ) has been shown to inhibit growth of normal breast epithelium, but promotes aggressive behaviors in breast carcinomas. Normal breast epithelia are exposed transiently to increased levels of TGFβ during involution. During radiation therapy for breast cancer, the breast epithelium is also exposed transiently to TGFβ.While TGFβ restricts growth of most normal breast epithelia, it may have divergent effects on progenitor cells. The CDβgeo cells have been shown to be enriched for mammary progenitor cells (Deugnier et al., 2006). Treatment of CDβgeo cells with TGFβ1 resulted in modest decreases in E-cadherin levels after 14 days (TD0). However, withdrawal of TGFβ and passaging the cells over 14 days resulted in transdifferentiation (TD14). In contrast to the parental CDβgeo cells, the TD14 cells had decreased expression of E-cadherin (3-fold, p<0.05) and increased levels of N-cadherin (4-fold, p<0.05). The TD14 cells also exhibited a striking increasing in motility and invasion of Matrigel (invasion index=3.7-fold), but there was no difference in proliferation rates between the CDβgeo and TD14 cells. The TD14 also were strongly tumorigenic when transplanted into cleared mammary fat pads (TE50=43 days). To determine if Smad signaling is required, Smad4 expression was knocked down using shRNA (ΔSmad4). The ΔSmad4 cells exhibited decreased expression of E-cadherin, but were not tumorigenic when transplanted into cleared mammary fat pads. The levels of Snail mRNA in CDβgeo cells were increased 3-fold following exposure to TGFβ1 treatment (TD14 cells). Levels of Snail were also increased (3.5-fold) in tumors derived from the TD14 cells. Although E-cadherin expression was reduced in ΔSmad4 cells, the levels of Snail remained equivalent to that in CDβgeo cells even after exposure to TGFβ1. These results demonstrate that transient exposure to TGF-β1 acts through Smad4-dependent signals to cause irreversible changes in the expression of Snail resulting in tumorigenic phenotype.

Citation Information: Cancer Res 2009;69(23 Suppl):A5.
Abstract A54: CnABP, a novel modulator of the Calcineurin-NFAT signaling pathway, is overexpressed in Wilms' tumors and promotes cell migration
Authors: Nguyen Alana H.T. , Beland Melanie , Gaitan Yaned , Bouchard Maxime
Published: May 2012

Wilms' tumor (WT) is a classical cancer type that arises from abnormal differentiation of kidney progenitor cells, and occurs at a frequency of 1 in 10,000 live births, accounting for approx. 90% of childhood kidney cancer. Abnormally high levels of Pax2 (paired-box protein 2), a key regulator of kidney development, have been observed in both WT and renal cell carcinoma, which correlate with proliferation and increased invasiveness. Therefore the misexpression of Pax2 and its target genes may play an important role in tumor initiation and/or progression. To test this, we screened for target genes of Pax2 by cDNA microarray in the embryonic kidney. We identified CnABP (Calcineurin A Binding Protein), a novel gene under Pax2 regulation. In situ hybridization indicates that CnABP coexpresses with Pax2 in the condensing mesenchyme, the abnormal differentiation of which gives rise to WT. Furthermore, expression analysis by quantitative PCR indicates that CnABP is overexpressed in more than 70% of Wilms' tumors. Interestingly, in the proportion of tumors with upregulated PAX2 expression, more than 80% also overexpress CnABP. We characterized CnABP as a membrane-anchored protein that primarily promotes cell proliferation and migration. Mediators of these activities were investigated by Yeast-two-hybrid and immunoprecipitation, which identified an interaction between CnABP and Calcineurin A, the catalytic subunit of a calcium-responsive serine/threonine phosphatase Calcineurin. We showed that CnABP modulates phosphatase activities of Calcineurin, which consequently inhibits calcium/Calcineurin-dependent NFAT nuclear translocation. We further demonstrated that the inhibition of NFAT nuclear localization results in reduced NFAT-specific transcriptional activity. Components of the Calcineurin complex have been reported as differentially expressed genes distinguishing recurrent from non-recurrent Wilms' tumors. This is in line with the evidence we presented, as CnABP is upregulated in Wilms' tumors and is shown to promote proliferation and migration.

Citation Information: Cancer Res 2009;69(23 Suppl):A54.
Abstract A58: Cdk6 association with a transcriptional coactivator indicates a potential role for this cyclin dependent kinase in regulating differentiation
Authors: Crary Jennifer I. , Grossel Martha J. , Hinds Phil W.
Published: May 2012

Cdk6 is a well characterized cyclin dependent kinase that regulates the transition from G1 phase to S phase during the cell cycle with the assistance of the D-type cyclins. Cdk6 is an oncogene and has long been considered to be functionally redundant to cdk4. Yet recent studies have shown functional discrepancies between the two kinases, with cdk6 often displaying distinct roles in development. In this study, a yeast two-hybrid analysis was used to identify potential binding partners for cdk6 from a human fetal brain library. We have confirmed the association with one designated protein, Eya2, via an in vitro GST pull-down assay. Eya2 is a mammalian homologue of the Drosophila gene eya, which is best known for its role as a co-activator involved in transcriptional regulation during eye development. Initial comparison of cdk4 and cdk6 in the GST pull-down assay indicate that cdk6 has a higher binding affinity for Eya2 than cdk4; interestingly, the yeast two-hybrid experiments failed to demonstrate an interaction between cdk4 and Eya2. Additional experiments are underway to confirm this interaction with several in vivo approaches that may further our understanding of mechanisms involved in cancer and development.

Citation Information: Cancer Res 2009;69(23 Suppl):A58.
Abstract A6: Dissecting mechanisms of RANKL dependency in osteosarcoma
Authors: Beristain Alexander G. , Molyneux Sam , Di Grappa Marco A. , Khokha Rama
Published: May 2012

Osteosarcoma is the most common bone tumor in adolescents, severely impacts quality of life, and ∼40% of patients die of metastases to lungs and liver. In an effort to study this poorly understood cancer, we have generated a novel transgenic mouse model of osteosarcoma (designated MOTO) through osteocalcin promoter-driven SV40 T-antigen expression. The MOTO model is 100% penetrant and recapitulates all critical features of the human disease including skeletal tumor distribution, radiology, histology, genomic instability and metastasis. Using genomic screens, we have identified the cytokine RANKL (Receptor Activator of Nuclear Factor kappa B Ligand), and its receptor RANK, to be aberrantly expressed in MOTO tumors and cell lines. Genetic studies using RANKL deficient MOTO mice reveal osteosarcoma to be broadly dependent on its expression. To further dissect the role of RANKL signaling in osteosarcoma, we have performed gain- and loss-of-function studies to determine if RANKL affects classic oncogenic cell parameters. Specifically, loss of RANKL in MOTO tumor cell lines inhibits invasion and soft agar colony formation, indicating its ability to contribute to tumorigenesis in a cell autonomous manner. To support this hypothesis, we are testing the effects of de novo RANK expression on the tumorigenic properties of osteoblastic cells. Together, these studies are dissecting the individual contributions of RANKL and RANK in controlling primary bone tumorigenesis.

Citation Information: Cancer Res 2009;69(23 Suppl):A6.
Abstract A67: Regulation of Eag1 expression by steroid hormones in cervical cancer cells
Authors: Rodriguez-Rasgado Jesus Adrian , Diaz Lorenza , Garcia-Becerra Rocio , Avila Euclides , Camacho Javier
Published: May 2012

Introduction: Ether à go-go 1 (Eag1) channels have oncogenic properties. This channel is widely expressed in human tumors. It has been shown that inhibition of its expression and/or activity reduces cellular proliferation. Some cancer-associated factors up-regulate Eag1 expression; including estradiol and the E6 and E7 human papilloma virus (HPV) oncogenes. On the other hand, calcitriol (the most active metabolite of vitamin D) has shown antiproliferative properties and it has been considered as a potential anticancer treatment. In this work, we studied Eag1 regulation by estradiol, tamoxifen, ICI 182 780, genistein and calcitriol in the cervical cancer cell line SiHa, that express E6 an E7 HPV oncogenes.

Methods: SiHa cells were cultivated with DMEM without phenol red and were treated with estradiol, tamoxifen, ICI 182 780, genistein or calcitriol during 48 hours. Total RNA was obtained with Trizol reagent, and cDNA was synthesized. Eag1 expression was evaluated by real time PCR. Eag1 expression was normalized against the housekeeping gene GAPDH. Using XTT colorimetric assay we evaluated the effect of calcitriol on SiHa cells proliferation.

Results: Calcitriol decreased cell proliferation after 5 days of culture. We also identified estrogen receptors α and β and VDR (vitamin D receptor) proteins by Western blot. Estradiol, antiestrogens, genistein and calcitriol down-regulated Eag1 mRNA expression. Concomitant treatment of SiHa cells with estradiol and calcitriol seems to potentiate such Eag1 down-regulation. On the other hand, SiHa cells expressed higher levels of ERβ than ERα.

Conclusions: Our data suggest that down regulation of Eag1 by calcitriol could be a new mechanism by which calcitriol reduce tumor cells proliferation. Together, estradiol, antiestrogens, genistein and calcitriol could be used as treatment for cancers that present ER pattern expression silimar to SiHa cells.

Citation Information: Cancer Res 2009;69(23 Suppl):A67.
Abstract A70: Oncogenic potential of multiple splicing isoforms of the prostate-cancer specific TMPRSS2-ERG gene fusion product
Authors: Zammarchi Francesca , Boutsalis George , Gerald William , Cartegni Luca
Published: May 2012

Genetic rearrangements are key events in cancer development, as seen by the recurrent chromosomal translocations characteristic of some hematological and mesenchymal tumors. It has been recently reported, however, that a majority of prostate cancer tumors appear to carry specific translocation, in which a transcription factor of the ETS (E26 transformation-specific) family (namely ERG, ETV1, ETV4, ETV5) is expressed under the control of TMPRSS2, a prostate-specific, androgen-dependent protease. In particular, the fusion of TMPRSS2 exon 1 to ERG exon 4 has been observed in approximately 50% of human prostate cases, suggesting a causative role in PC development, but its specific functions remain unclear. This is further complicated by the presence of ERG splicing variants, and by the use of alternative promoters or polyadenylation sites in both the normal and fusion contexts, which could generate over 50 different isoforms. Interestingly, the presence of specific isoforms seems to correlate with tumor aggressiveness.

ERG is already known to be involved in oncogenic fusion, such as EWS-ERG fusion in Ewing sarcoma, which activates targets involved in proliferation, metastasis and angiogenesis, suggesting that the androgen-dependent over-expression of ERG following the TMPRSS2 fusion could be a driving mechanism of prostate carcinogenesis.

We have identified multiple novel splicing isoforms of both the wild-type ERG gene and the TMPRSS2:ERG fusion gene in prostate tumors and we are currently investigating their respective biological roles and mechanism of action in order to determine their function and oncogenic potential in vitro and in vivo.

Citation Information: Cancer Res 2009;69(23 Suppl):A70.
Abstract A72: Stat3 is a negative regulator of intestinal tumor progression in APCmin mice
Authors: Musteanu Monica , Blaas Leander , Mair Markus , Casanova Emilio , Kenner Lukas , Poli Valeria , Eferl Robert
Published: May 2012

The transcription factor Stat3 has been considered to promote progression and metastasis of intestinal cancers. We investigated the role of Stat3 in intestinal tumors using mice with conditional ablation of Stat3 in intestinal epithelial cells (Stat3ΔIEC). In the APCmin mouse model of intestinal cancer, genetic ablation of Stat3 reduced the multiplicity of early adenomas. However, loss of Stat3 promoted tumor progression at later stages leading to formation of invasive carcinomas which significantly shortened the lifespan of Stat3ΔIEC APCmin/+ mice. Interestingly, loss of Stat3 in tumors of APCmin/+ mice had no significant impact on cell survival and angiogenesis but promoted cell proliferation. A genome-wide expression analysis of Stat3-deficient tumors suggested that Stat3 negatively regulates intestinal cancer progression via the cell adhesion molecule Ceacam1. Therefore, detrimental effects on tumor progression have to be considered upon therapeutic targeting of the Stat3 signaling pathway in intestinal cancer.

Citation Information: Cancer Res 2009;69(23 Suppl):A72.
Abstract A8: Role of the WWOX gene in non-small cell lung cancer
Authors: Buyru Nur , Baykara Onur , Demirkaya Ahmet , Kaynak Kamil , Toker Alper , Tanju Serhat
Published: May 2012

Lung cancer is the most common cause of cancer-related deaths worldwide and like many other cancers is affected by different genetic, epigenetic and environmental factors. The WWOX gene is a tumor supressor gene located on 16q23.3–24.1. It has been shown that it loses its function due to differences in genetic and epigenetic mechanisms.

In this study we investigated whether the presence of WWOX gene mutations in lung cancer. The entire coding exons and flanking intronic sequences of the gene were analysed by PCR amplification from the genomic DNA and direct sequencing in 50 patients with non-small cell lung cancer (NSCLC). WWOX gene mutations were detected in five out of 50 primary tumors (10%). One of these mutations was an insertion in exon 4 at the third nucleotide of codon 120. This mutation was observed in 2 patients and leads to a frameshift creating a stop codon at codon 133. The second mutation was an insertion at position 2583 which also causes a shift in the reading frame. Finally, heterozygous C→G transitions on position 2807 at the first nucleotide of codon 282 (exon 8) were identified in 2 patients. This mutation affecting codon 282 leads to the substitution of alanine instead of proline. We also observed a previously defined G to A polymorphism at the nucleotide 2196 in homozygous and heterozygous form in 20 and 5 patients, respectively. Additionally we detected 12 different intronic substitutions. In addition to inactivating mutations target genes may also be silenced epigenetically. Therefore, besides mutation screening we also analyzed the methylation profile of the promoter region of the WWOX gene in tumor tissue and adjacent non-cancerous tissue samples by bisulphite modification and methylation-specific PCR. Promoter methylation was detected in 76% of the tumor tissue samples. While 20% of the samples displayed partial methylation 56% were completely methylated. In the adjacent normal tissue samples partial and complete methylation frequencies were 6% and 58%, respectively. Methylation frequency was significantly higher in the tumor tissue samples than in normal tissue samples (p=0.014).

The high frequency of mutations and epigenetic modifications suggest that the WWOX gene may play a significant role in the etiology of NSCLC.

Citation Information: Cancer Res 2009;69(23 Suppl):A8.