bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 490: Different localization of epidermal growth factor receptor affects the response of Cetuximab in the irradiated NSCLC cell lines
Authors: Ryu Seung-Hee , Yang Youn-Joo , Moon Soo Young , Oh Jeong-Yoon , Song Si Yeol , Kim Jong Hoon , Choi Eun Kyung , Ahn Seung Do , Lee Sang-wook
Published: February 2011

The overexpressed epidermal growth factor receptor (EGFR) mediates resistance of tumor cells to chemo- and radiotherapy resulting from the induction of DNA repair by DNA-PK activation. The blockage of EGFR by a specific antibody (cetuximab) or tyrosine kinase inhibitor can inhibit the nuclear import of EGFR and consequently increase cellular sensitivity to ionizing radiation. Even though cetuximab combined with radiation showed encouraging response rates in locally advanced head and neck cancer, there are negative results in a recent trials comparing cetuximab and chemotherapy to chemotherapy alone. Thus it is critical to preselect patients based on their biologic factors to predict sensitivity for combination of cetuximab and radiation. The human non-small cell lung cancer (NSCLC) cell lines, A549 and H1299 which contain wild type EGFR, were used to demonstrate the different response to cetuximab in the combination with radiotherapy. Localization of EGFR was detected by confocal microscope and radiosensitivity was measured by clonogenic assay. Treatment of cetuximab inhibited colony formation in a dose-dependent manner in A549 cell line, while did not affect that in H1299 cell line. Microscopic images revealed that EGFR localized in cytosolic fraction especially around golgi body in H1299 cell line instead of the localization in membrane fraction in A549 cell line. After irradiation nuclear EGFR was detected in A549 cell line, however EGFR did not translocate to nucleus in H1299 cell. Even though decreased EGFR expression both A549 and H1299 cell lines was detected in the combination with cetuximab and radiation, radiosensitivity was increased only in A549 cells, not in H1299 cells. Our data indicated that localization of EGFR is evident marker to determine the efficacy of cetuximab and may relate sensitivity/resistance of cells treated cetuximab in the combination with radiotherapy. We suggest that EGFR status can be used for preselection of patients to predict treatment sensitivity in the combination therapy. *Grant Support : This study was supported by a grant (A090401) of Korea Healthcare Technology R&D Project, Ministry for Health, Welfare & Family Affairs, Republic of Korea.

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 490.
Abstract 4905: Quantitative high resolution detection of O6-MGMT methylation in routinely processed human glioblastoma specimens
Authors: Lehmann Ulrich , Weidner Jenny , Albrecht David , Schipper Elisa , Brandis Almuth , Kreipe Hans H.
Published: February 2011

Hypermethylation of the DNA repair gene 06-Methylguanosinemethyltransferase (06-MGMT) is an important prognostic and predictive marker for the management of glioblastoma patients. However, assessment of O6-MGMT gene methylation is not yet standardized and a comprehensive comparison of different methods described in the literature is missing.

Therefore, the methylation status of the MGMT gene was analyzed in a large series of glioblastoma specimens, WHO grade IV (n = 92) using nested conventional methylation-specific PCR (MSP), real-time PCR using either SybrGreen or a sequence specific probe for detection and pyrosequencing. All specimens were routinely processed formalin-fixed paraffin-embedded tumor biopsies. For a subset of samples (n = 30) three different protocols for the treatment of genomic DNA with bisulfite were compared. As a control, 20 brain biopsies from 10 patients without glioblastoma were analyzed. MGMT mRNA level was measured using quantitative real-time RT-PCR in n = 39 samples and the mRNA expression levels were correlated to the methylation status of these samples.

The three different bisulfite protocols did not show statistically significant differences concerning quality and yield of recovered DNA. A region in the MGMT promoter in which many published primers bind, displayed constitutively elevated methylation levels also in healthy brain tissue samples, thereby leading to false-positivity under many circumstances. Nested MSP and pyrosequencing correlate fairly well in scoring samples as “methylated” versus “unmethylated”, but a considerable number of samples scored “methylated” by nested MSP (n = 7) displayed only background methylation when analyzed by pyrosequencing. Only methylation levels measured by pyrosequencing correlated with mRNA expression levels (r2 = 0.32, p = 0.0002). Also, threshold definition was much more straightforward and easier to standardize using pyrosequencing. Real-time PCR based methods did not correlate very well with nested PCR or pyrosequencing.

In conclusion, pyrosequencing is the most reliable methodology for methylation analysis of O6-MGMT gene methylation analysis in routinely processed glioblastoma specimens.

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 4905.
Abstract 4911: HPV-independent methylation of specific TGF-B pathway-relevant genes in head and neck squamous cell carcinomas associated with prior radiation therapy and exposure to alcohol and tobacco
Authors: Bennett Kristi L. , Romigh Todd , Lee Walter , Lamarre Eric , Zhang Xiatong , Seth Rahul , Scharpf Joseph , Hunt Jennifer , Eng Charis
Published: February 2011

Head and neck squamous cell carcinoma (HNSCC) is an aggressive malignancy with more than half a million people being diagnosed with the disease annually. Therefore, it is critical to identify early detection markers, such as DNA methylation, for more timely diagnosis. In a recent study, we identified five tumor suppressive genes (IRX1, EBF3, SLC5A8, SEPT9, and FUSSEL18) as frequently methylated in HNSCC biopsies using a global methylation analysis via restriction landmark genomic scanning (RLGS). Interestingly, we found these candidates are all potentially involved in the TGF-B signaling pathway, which is often disrupted in HNSCC. Therefore, we sought to determine coordinated epigenetic silencing of these candidate genes in HNSCC as potential key disruptors of TGF-B signaling, which could ultimately result in HNSCC progression. Through immunoprecipitation studies, all five of the investigated candidate genes were found to interact with components of the TGF-B pathway. Also, overexpression of SLC5A8, EBF3, and IRX1 resulted in decreased mitotic activity and increased apoptosis. These findings are significant because they reveal a set of genes that interact with components of the TGF-B pathway, and their silencing via methylation in HNSCC results in coordinated decrease in apoptosis, increased proliferation, and decreased differentiation. We went on to define the methylation of these genes in relationship to HPV16-positivity, radiation therapy, and alcohol and tobacco exposure. By utilizing the available clinical information linked to the patient specimens, we found a strong association between promoter methylation of FUSSEL18, IRX1, and EBF3 and prior radiation therapy (P < 0.0001) irrespective of HPV status. Also, promoter methylation of FUSSEL18 and SEPTIN9 were found to correlate significantly with exposure to alcohol and tobacco (P = 0.021). Importantly, in this study we preliminarily show a trend between HPV16 positivity and specific target gene hypermethylation of IRX1, EBF3, SLC5A8, and SEPT9. Equally notable and independent of HPV status, hypermethylation of the promoters of a subset of these genes in recurrences especially in the setting of prior radiation or in the setting of alcohol and tobacco use might help guide adjunctive inclusion or exclusion or epigenetic therapy.

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 4911.
Abstract 4924: Gene-specific hyper and hypomethylation in follicular lymphoma progression
Authors: Saggese Matilde , Thodima Venkata , Heguy Adriana , Chan Timothy A. , Chaganti Raju S. K.
Published: February 2011

Epigenetic mechanisms involved in the origin and transformation of indolent lymphomas to aggressive forms are poorly understood. Based on the comparison of methylation states of follicular lymphoma (FL) pre and post- transformation, it has been suggested that DNA methylation changes occur during the early stages of lymphomagenesis. To obtain a more complete understanding of methylation aberrations responsible for progression and transformation of FL, we analyzed 20 untreated FL biopsies (10 low grade, 10 high grade), 5 diffuse large B cell lymphoma (DLBCL) biopsies, and 5 benign reactive lymph node (RLN) biopsies by using methyl-DNA immunoprecipitation coupled with analysis with Agilent 244K human promoter ChIP-on-chip microarrays. Our analysis revealed statistically significant differences in methylation status for 120 genes between FL samples and RLN samples and for 23 genes between low and high grade FL samples. To investigate the biological significance of these findings, we used DAVID functional annotation clustering algorithms and Gominer software. We found that the most methylation-enriched functional groups were comprised of genes with roles in cell cycle control, adhesion and motility, transcription regulation, homeobox, and developmental genes. In order to validate the DNA methylation differences of the significant gene regions, we utilized the MassARRAY EpiTYPER assay. Our data identified 13 genes (CYP26B1, GRM7, HS3ST2, DOCK5, GRB10, SST, TAC1, PALM2-AKAP, FLJ40125, ST6GAL2, WDR35, NCAM1, SATB2) with increased levels of DNA methylation in FL samples compared to controls; 3 of these genes (ST6GAL2, WDR35, NCAM1) exhibited increased methylation in high grades FLs and 1 gene (SATB2) in low grades FLs. We are currently investigating gene specific DNA hypomethylation in low versus high grade FL and FL versus DLBCL. Our data are the first to characterize the global changes in methylation that occur in FL progression. Our findings suggest that high grade FL are associated with hypermethylation of a specific subset of genes compared to low grade subtypes. Ongoing efforts include investigating the functional relevance of these methylation changes by treating, in vitro, FL cell lines with demethylating agents to evaluate changes in gene expression levels and correlating with phenotype alterations.

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 4924.
Abstract 4952: Study of mouse tumor models with an IRDye 800CW SNAP-tag imaging probe
Authors: Zhang Aihua , Sun Luo , Kovar Joy , Gong Haibiao , Oliver D. Michael , Correa Ivan , Russello Salvatore , Noren Christopher , Xu Ming Qun
Published: February 2011

Non-invasive optical analysis of molecular targets inside living animals has become an important tool for disease progression and treatment assessment. One of the essential elements of molecular imaging is the development of specific, sensitive imaging contrast agents to investigate these biological processes. The use of longer wavelength dyes, i.e. near-infrared (NIR) dyes, presents distinct advantages over inherently fluorescent protein tags, including reduced autofluorescence background and increased sensitivity in imaging small animals. In addition, the adoption of efficient and specific labeling techniques is a key step in the generation of protein-based fluorescent imaging agents. In the present study, we explore the versatility of a self-labeling protein termed SNAP-tag, derived from human O6-alkylguanine-DNA alkyltransferase, in tagging polypeptides with a near-infrared dye, IRDye® 800CW. In cell-based assays, an IRDye 800CW conjugated SNAP-tag successfully labeled cells transiently transfected with a pSNAP-ADRB2 plasmid. For in vivo experiments, a synthetic epidermal growth factor receptor (EGFR)-binding ligand was used to construct an IRDye 800CW-EGFR-SNAP-tag. Following injection of the agent into nude mice bearing EGFR-overexpressing A431 xenografts, tumors were clearly visualized with a Pearl® Imaging system. Furthermore, the specificity of binding was demonstrated in competition experiments with unlabeled EGF. Similar probes labeled with visible fluorophores were used to image EGFR-expressing cultured cells by confocal microscopy. This fluorescent imaging system using a self-labeling protein tag and highly sensitive organic dyes provides versatile tools for cancer research, drug discovery and small animal imaging.

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 4952.
Abstract 4959: Multispectral imaging of chromogenic multiplex IHC assays as an alternative to fluorescent methods
Authors: Kerfoot Christopher A. , Girees Sherif , Dauffenbach Lisa M.
Published: February 2011

Detection and analysis of two or more co-localized antigens by light microscopy has been hindered by the difficulty in discerning and quantifying overlaying chromogens. Performing multi-analyte immunohistochemistry has many potential benefits and applications in the field of drug target evaluation and patient screening, including: 1) determination and quantification of immunoreactivity co-localization; 2) evaluation of spatial relationships between immunoreactive cell types; 3) signal transduction pathway activation studies; 4) reduction in tissue depletion; and 5) decreased reagent utilization. Multiplex immunofluorescence is not widely used as an analytical technique for clinical samples due to its lower sensitivity than chromogenic methods and concerns about autofluorescence in formalin-fixed tissue. Through the use of multispectral imaging, the absorption pattern of overlapping chromogens can be spectrally unmixed into quantitative individual dye components. This process works accurately when chromogens are developed to a lower intensity than typical for most standard pathology laboratories. The chromogens must be developed to a point where they do not scatter light or block subsequent chromogenic development. The current study was performed to define the accuracy of spectral unmixing of chromogens and evaluate whether interference existed between chromogens. Stained samples were imaged using multispectral imaging, and quantitative grayscale images for each analyte were created by spectral unmixing. The grayscale images were analyzed using image analysis software to determine the overall expression levels of each analyte. Development with Vulcan Red followed by DAB produced little interference, while high density development with DAB decreased the subsequent Vulcan Red staining intensity. Vulcan Red, DAB and hematoxylin unmixed properly and quantitatively. In summary, when multiplexed chromogenic IHC assays were developed using methods optimized for multispectral imaging, the technique demonstrated excellence performance characteristics without suffering from drawbacks commonly associated with fluorescence-based techniques.

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 4959.
Abstract 4968: Modes of co-factor recruitment by an oncogenic fusion protein
Authors: Schwentner Raphaela , Kauer Maximilian O. , Bilke Sven , Jug Gunhild , Walker Robert L. , Meltzer Paul S. , Kovar Heinrich
Published: February 2011

Ewing's sarcoma (ESFT) is a highly aggressive pediatric cancer of the bone and characterized by the chimeric ETS transcription factor EWS-FLI1 as a result of a chromosomal translocation. By whole genome gene expression and ChIP-seq analyses we obtained evidence that EWS-FLI1 directly regulates E2F target genes and possibly cooperates with E2F transcription factors. Furthermore, several E2F factors were themselves inferred to be direct targets of EWS-FLI1. To unravel the hierarchical structure of this EWS-FLI1 transcriptional network module we performed luciferase reporter gene assays in combination with DNA motif mutation analyses. The promoter regions of nine candidates for direct target genes of EWS-FLI1 and E2F factors were chosen for in-depth analysis. Corroborating the transcriptomic data, promoter activity of all 9 genes showed reduced levels upon knockdown of EWS-FLI1. The strongest modulatory effect of EWS-FLI1 silencing was observed for the E2F3 promoter, and mutation of a single ETS binding site lead to a comparable reduction of luciferase activity as knockdown of EWS-FLI1. These results validate E2F3 as a directly EWS-FLI1 regulated gene in Ewing's sarcoma. For the other EWS-FLI1 target candidates mutation analysis lead to more complex results. In the RAD51 promoter simultaneous mutation of two ETS binding sites was necessary to obtain a significant reduction of luciferase activity. Furthermore, mutation of a single E2F binding site abolished the effect of EWS-FLI1 knockdown. In promoters of two other candidates, GEMIN4 and ATAD2, mutation of the ETS site resulted in only a moderate reduction of reporter activity, and additional destruction of an E2F binding site was required to achieve a significant decrease in promoter activity. Our results imply that EWS-FLI1 uses several modes of interaction with E2F factors to control common target genes. First, EWS-FLI1 directly activates E2F3 and other E2F factors which, in turn, co-operate with EWS-FLi1 on target gene promoters in a feed-forward loop. Second, results for the RAD51 promoter are compatible with a mechanism where a repressive E2F factor is exchanged for an activating E2F factor via binding of EWS-FLI1. Finally, EWS-FLI1 might have a more indirect role in the regulation of ATAD2 and GEMIN4 where most of the transcriptional effect seems to be exerted by E2F factors.

Acknowledgement: This study was funded by the 6th framework program of the European Commission, (“E.E.T. Pipeline” contract LSHC-CT-2006-037260) and by the Austrian genome research program “GEN-AU Ch.I.L.D.“ (GZ 200.136/1 - VI/1/2005). R. Schwentner is a recipient of a DOC-fFORTE-fellowship of the Austrian Academy of Sciences.

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 4968.
Abstract 4970: Complex regulation of RIP140 gene expression by E2F transcription factors
Authors: Docquier Aurelie , Harmand Pierre-Olivier , Fritsch Samuel , Badia Eric , Fajas Lluis , Augereau Patrick , Cavailles Vincent
Published: February 2011

We recently showed that RIP140 (Receptor Interacting protein of 140 kDa), a regulator of nuclear receptor activity, was involved in the control of cell proliferation and able to repress E2F1 activity. We now demonstrate that RIP140 and E2F1 comprise a novel transcriptional regulatory loop. We first observed that RIP140 mRNA levels increased during the G1/S and G2/M transitions and were reduced in tissues from E2F1 null mice. Furthermore, we identified the RIP140 gene as a transcriptional target of E2F1, showing, by transient transfection, that overexpression of E2F1/DP1 strongly increased transcription of the RIP140 promoter. Bioinformatic analysis identified several binding sites for E2Fs in the proximal promoter region and we evidenced the direct binding of E2F1/DP1 on these sequences by gel shift analysis. However, site directed mutagenesis of these elements only modestly affected transactivation of the RIP140 promoter by E2F1/DP1, thus suggesting an indirect recruitment of E2Fs on the promoter. This was strengthened by the observation that overexpression of E2F1, 2 or 3 alone was very potent in increasing transcription from the RIP140 promoter. Moreover, overexpression of DP1 produced a negative effect on this regulation which was also strongly repressed by RIP140 itself. Altogether, these results revealed a complex control loop between E2Fs and RIP140 and the molecular mechanisms underlying these atypical regulations are currently under investigation.

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 4970.
Abstract 4978: FOXP3 up-regulates p21 expression in breast cancer: Evidence for tumor suppressor
Authors: Liu Runhua , Wang Lizhong , Katoh Hiroto , Zheng Pan , Liu Yang
Published: February 2011

FOXP3 is the first X-linked breast cancer suppressor gene that works at least in part by repression of oncogenes, including HER-2 and SKP2. Since FOXP3 is also known as a transcription activator, we investigated the possibility that FOXP3 may induce expression of tumor suppressor genes. p21 loss has been implicated in conferring oncogenic activity to known tumor suppressor gene KLF4 and cancer drug tamoxifen. Regulators of p21, therefore, play critical roles in tumorigenesis. Here, we report that X-linked tumor suppressor FOXP3 is essential for p21 expression in normal epithelia and that lack of FOXP3 is associated with p21 down-regulation in breast cancer samples. Among 62 primary breast cancer samples tested, a strong association between FOXP3 down-regulation and lack of p21 was observed. Moreover, siRNA silencing of FOXP3 in primary human mammary epithelial cells leads to a dramatic reduction in expression of p21, which demonstrates an important role of FOXP3 in maintaining expression of p21 in normal breast epithelial cells. In breast cancer cell line MCF-7, an inducible expression of FOXP3 is associated with induction of p21. Since silencing p21 reduced the efficacy of FOXP3-mediated growth inhibition, the induced expression of p21 contributes to growth inhibition by FOXP3. A specific FOXP3 binding site in the intron 1, which is associated with acetylated histone H3, is essential for p21 induction by FOXP3. FOXP3 specifically inhibited binding of histone deacetylase 2 (HDAC2) and HDAC4 to the site and increased local histone H3 acetylation. Short hairpin RNA silencing of either HDAC2 or HDAC4 is sufficient to induce p21 expression. Our data provides a novel mechanism for transcription activation by FOXP3 and a genetic mechanism for lack of p21 in a large proportion 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 4978.
Abstract 4979: The role of Tel in repression of BCL-XL transcription in tumor cells
Authors: Cao Xiaobo , Peddaboina Chander , Jupiter Daniel , Zhang Lidong , Littlejohn James , Rascoe Philip , Smythe Roy
Published: February 2011

Study of Bcl-xl and hepatocyte growth factor (HGF) receptor c-Met demonstrates that ETS transcription factors influence the transcriptional control of Bcl-xl in multiple tumors. The phosphorylation of Tel, a transcriptional repressor in the ETS family, by activated c-Met is required to increase Bcl-xl transcription in addition to activation of ETS2, a transcriptional activator.

Our data indicate that stimulation by HGF causes the activation of ERK and leads to the nuclear export of Tel. In order to determine whether ERK is responsible for the phosphorylation of Tel after HGF stimulation, physical interaction between Tel and ERK was detected using I.P. (Tel)-western blotting (ERK). To determine if blocking MAP kinase could prevent Tel's phosphorylation after HGF exposure, we again used I.P. Western to compare the phosphorylation of Tel under specific ERK kinase inhibitors, after HGF exposure, to the phosphorylation of Tel in the absence of inhibitors, after HGF exposure. A His-Tel 213mutant (phosphorylation sites mutants) was created. The Tel 213 mutant did not undergo phosphorylation induced by c-Met activation, as demonstrated by I.P. Western blot. Nuclear localization of mutated His-Tel was detected under HGF/EGF exposure via fluorescent microscopy using FITC labeled anti-his antibody, indicating that phosphorylation of Tel is also required for nuclear export. A luciferase reporter assay shows that the transient expression of Tel 213 mutant in cancer cell lines results in further repression of Bcl-xl promoter activity. Indeed, Tel 213's repressive function is independent of c-Met stimulation, as determined by comparing with wild type Tel cDNA transfected cells. Consequently, Tel 213 transfection results in much stronger reduction in Bcl-xl mRNA and protein expression levels in the presence of HGF, as compared to transfection with wild-type Tel cDNAs in the presence of HGF. Furthermore, a CHIP assay demonstrates that the activation of c-Met reduced the binding of wild-type Tel to the Bcl-xl promoter, while the binding of Tel 213 to bcl-xl promoter was not affected. Finally, transfection of Tel 213 leads to increased cell proliferation inhibition and apoptosis.

Taken together, these findings are supportive of a role for Tel, through a c-Met aixs, as an inhibitor of Bcl-xl transcription.

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 4979.
Abstract 4983: Galectin-3 regulates p21 expression through negatively regulating its degradation
Authors: Wang Yi , Nangia-Makker Pratima , Raz Avraham
Published: February 2011

Galectin-3 (Gal-3), a carbohydrate-binding protein, has been demonstrated to be involved in multiple biological events such as cell adhesion, cell migration, cell proliferation, apoptosis and so on. p21, originally described as a inhibitor of cyclin-dependent kinases (CDK), has been shown to have anti-apoptotic activity as well as regulation of cell proliferation. Since both of these two proteins play important roles in cancer and some of their functions overlap, we assumed that there may be some relationship between Gal-3 and p21. Here, we first reported that Gal-3 positively regulates p21 expression in cancer cells, and this effect works at protein degradation level. Over-expression of Gal-3 in Gal-3 null human prostate cancer cell line LNCap and breast cancer cell line BT549 inhibited p21 degradation. Moreover, Gal-3 knockdown in Gal-3 expressing prostate cancer cell line DU145 potentiated p21 degradation, suggesting that Gal-3 negatively regulates p21 degradation in cancer cells. Furthermore, our data showed that Gal-3 negatively regulated proteasomal turnover of p21 in an ubiquitin-dependent way. In this study, we also showed for the first time the direct interaction between Gal-3 and p21. This study suggested that p21 may serve as the downstream molecule of Gal-3 signal transduction pathway to mediate some of Gal-3 functions, and it helps to understand further the mechanisms for Gal-3 functions.

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 4983.
Abstract 4997: The expression PHLPP is regulated by the mTOR-P70S6K signaling pathway
Authors: Liu Jianyu , Gao Tianyan
Published: February 2011

PHLPP is a novel family of protein phosphatases serving as negative regulators of Akt by directly dephosphorylating the kinase. Our previous studies have indicated a tumor suppressor role of PHLPP in human colon cancer. However, the regulation of PHLPP expression in cancer cells remains unknown. Here, we report that the expression of PHLPP protein is regulated by the mTOR-p70S6K pathway in colon and breast cancer cell lines. Treating cells with rapamycin, an inhibitor of mTOR, induces a marked decrease of PHLPP protein expression, and this rapamycin-mediated down-regulation of PHLPP expression is blocked by overexpression of a constitutively active mutant of p70S6K. Stable knockdown of TSC2, a negative regulator of mTOR, using lentiviral-based RNAi results in an increase of PHLPP expression, while knockdown of mTOR decreases the expression level of PHLPP protein. In addition, knockdown of 4E-BP1, a translation initiation inhibitor and a downstream effector of mTOR, results in an increase of PHLPP expression and resistance to rapamycin or Torin1 induced decrease of PHLPP expression in cells. Furthermore, amino acid starvation of cells, which inactivate the mTOR signaling, decreases PHLPP expression, and this is reversed by resupplying amino acids. Control experiments show that the rate of PHLPP degradation was not alerted significantly by rapamycin treatment or knockdown of TSC2. The real-time RT-PCR analysis reveals that the mRNA level of PHLPP remains constant in the TSC2 and mTOR knockdown or rapamycin treated cells. Taken together, these results suggest that the translation of PHLPP proteins is controlled by mTOR. Our studies identify a compensatory feedback regulation of Akt signaling in which the activation of Akt is negatively regulated by up-regulation of PHLPP protein in an mTOR-dependent manner.

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 4997.
Abstract 4999: Glutamate receptors in cancer: Stable expression of GluR6 induces growth arrest and senescence in hepatocellular carcinoma HCC 7703 cells
Authors: Kaur Gurpreet , Pilidis Konstantinos G. , Zhawar Vikramjit K. , Kaur Gursurinder P. , Athwal Raghbir S.
Published: February 2011

Applying a functional strategy, we identified a gene, SEN6A, located at 6q21, which restores senescence in breast tumor cells. Human genome database search identified SEN6A as GluR6, a kainate type - ionotropic glutamate receptor gene. Kainate receptors constitute a family of ionotropic glutamate receptors that plays a role in the rapid synaptic transmission in the central nervous system. Glutamate receptors have been implicated in several pathophysiological conditions including Epilepsy, Schizophrenia and Parkinson's disease. We cloned the GluR6 gene by RT-PCR and investigated the structure, splicing pattern and its role in the cell senescence and cancer. We show that GluR6A, a splice variant is specifically expressed in brain, and four other variants; namely, GluR6B, GluR6C, GluR6D and GluR6E are expressed in other tissues and fibroblast cell lines. Genomic analysis and cloning of the sequence preceding the transcribed region led to the identification of two tissue specific promoters designated as neuronal promoter Pn and non-neuronal promoter Pnn. GluR6 is expressed in most tissues and its expression is elevated in late passage cells, relative to young human cells. The gene is either turned off or expressed at a reduced level in a large number of tumor cell lines. The chromosomal location of GluR6 at 6q21, a LOH hot spot in multiple cancers, points to its potential role in the etiology of cancer. The analysis for mutations in 22 cancer cell lines revealed a point mutation in exon 15 and deletion of exon 6 in two hepatoma cell lines.

Open reading frame of GluR6 was cloned in a retroviral expression vector, in frame with EGFP tag, where EGFP is added after the signal peptide. In this vector GluR6-EGFP gene is expressed as a single transcript, from a CMV promoter, along with puromycin gene for selection. Ecotopic expression of GluR6 (variants A-E) induces growth arrest and senescence in hepatoma HCC7703 cells, and induce premature senescence in normal fibroblasts. Clones of GluR6 transfer cells display doubling time of 72-96 hours that gradually increases to 3-4 days ending in complete growth arrest and senescence. In contrast the transfer of empty vector or a construct carrying truncated GluR6 gene did not affect the growth of HCC7703 or normal cells. These result show that GluR6 is involved in the cascade of signaling circuits that govern cell growth and differentiation. Future studies are designed to investigate signaling pathways responsive to the expression of GluR6 gene.

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 4999.
Abstract 5000: Epigenetic de-silencing of Glutathione s-transferase pi by tumor suppressive maspin
Authors: Li Xiaohua , Heath Elisabeth , Sakr Wael A. , Sheng Shijie
Published: February 2011

Abstract: Loss of expression of the GST pi isoform due to DNA hypermethylation-mediated epigenetic silencing is one of the most common molecular events in precancerous and cancerous lesions of human prostate. Our previous studies demonstrated that tumor suppressive maspin, an endogenous inhibitor of histone deacetylase 1, could up-regulated GST activity and reduced reactive oxygen species (ROS) generation in response to oxidative stress treatment. We confirmed that maspin restored GST pi expression in maspin transfected prostate cancer cell line DU145. Conversely, maspin knocking down by siRNA in PC3 cells led to reduced GST pi expression. We further investigated the possibility that the epigenetic regulation by DNA-methylation and histone deacetylation were coupled dynamic processes that HDAC inhibition by maspin alone was sufficient to reverse the DNA-methylation silencing effects. Using methylation-specific polymerase chain reaction (M-PCR), DNA demethylation in GST pi promoter was detected only in maspin-expressing cells. Chromatin immunoprecipitation (ChIP) assay showed that a significant higher level of acetylated histone 3 was associated with the GST pi promoter DNA complex. In the same DNA complex from maspin transfected cells, less DNA methylation transferase (DNMT1) and methyl-CpG-binding domain (MBD2/3) proteins were detected as compared to that from the mock control cells. It was noted that in DU145-derived transfected cells, maspin simultaneously restored GST pi expression and sensitized tumor cells to drug-induced apoptosis, while overexprssion of GST pi alone in LNCaP prostate cancer cells conferred resistance to oxidative stress in cell death, HIF-1α induction, and phosphorylation of histone 2A.X. These data suggest that, as an epithelial-specific HDAC1 inhibitor, maspin may function to maintain the epithelial tissue homeostasis through balancing the GST pi-dependent detoxification and HDAC1 inhibition-dependent apoptosis sensitivity.

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 5000.
Abstract 5023: Down-regulating interferon regulatory factor-1 by siRNA promotes endurance to fulvestrant in sensitive breast cancer cells partially via Ras/Raf/MEK/ERK pathway
Authors: Ning Yanxia , Riggins Rebecca B. , Shajahan Ayesha N. , Zwart Alan , Clarke Robert
Published: February 2011

AIM: Interferon Regulatory Factor 1 (IRF1) is a tumor suppressor gene that has an antitumor role in some cancers. In this study, we showed that reducing IRF1 expression by siRNA promotes resistance to Fulvestrant in MCF7/LCC1 cells at least partly by acting though the Ras/Raf/MEK/ERK pathway. METHODS: After transfection with IRF1 siRNA by Amaxa Nuclofection, the MCF7/LCC1 cells were treated with 100 nM Fulvestrant for 2 days for monitoring gene expression, and for 3 and 6 days for apoptosis and cell proliferation assays, respectively. Five μM U0126 or 10 μM PD98059, the inhibitors of MEK, were used when necessary. Transactivation of IRF1, Bcl-2, Cyclin D1, and ERE were detected by luciferase assays after transfection with the appropriate reporter plasmid. Caspase 7, 8 and 9 activities were detected by cleavage of their respective substrates. RESULTS: After transfected with IRF1 siRNA, the mRNA and protein levels of IRF1 expression were greatly reduced in MCF7/LCC1 cells. Knocking-down IRF1 expression resulted in increased proliferation, increased S-phase ratio, and decreased apoptosis of MCF7/LCC1 cells compared to mock or control siRNA groups. mRNA and protein levels of Bcl-2, Bcl-w, Bcl-xL, Survivin and CyclinD1 were increased by IRF1 siRNA transfection. Luciferase assays showed that the Bcl-2 P2 (but not P1) promoter is inhibited by IRF1 siRNA transfection. At same time, the activities of Caspase 7 and 8 cannot be increased by Fulvestrant treatment after IRF1 siRNA transfection. Knocking down IRF1 expression partially restores Fulvestrant's inhibition of ERE-drive transcription. Total and phosphor-ER α expression were reduced by IRF1 siRNA transfection. Further, we found that the phosphor-ERK1/2 expression levels were increased by IRF1 siRNA transfection. Inhibition MEK with inhibitors could partially reverse the effect of IRF1 siRNA transfection on proliferation via inhibiting the effect of cell cycle other than apoptosis. Using the inhibitors has no effect on the expression of ER α and the transcriptional activity of ERE. CONCLUSIONS: Knocking down IRF1 expression promotes resistance to Fulvestrant in MCF/LCC1 breast cancer cells partially by increasing the expression of phosphor-ERK1/2. The reduced of ERα expression and increased transcriptional activity of ERE has also involved in the mechanism.

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 5023.
Abstract 5031: Combination inhibition of the FAK-p53 and MDM2-p53 interactions suppressed proliferation in mesothelioma
Authors: Ou Wenbin , Fletcher Jonathan A.
Published: February 2011

Mesothelioma is a chemotherapy-resistant neoplasm, as is evident in the dismal survival for patients with this asbestos-associated disease. Conventional chemotherapies and radiation therapy have only limited efficacy, and improved survival will require development of novel and more effective pharmacological interventions. Although p53 genomic mutations are uncommon in mesothelioma, we hypothesized that functional p53 inactivation (resulting from NF2-dysregulated FAK activation) might be a frequent event. Using proteomic methods, with tandem mass spectrometry analyses of mesotheliomas after double immuno-affinity purifications for p53 and phosphotyrosine, we find that FAK is consitutively activated in 10 of 10 mesothelioma cell lines and 9 of 9 mesothelioma surgical specimens, and associated with p53 in 6 of 6 mesothelioma cell lines. In four mesotheliomas with non-mutant p53, FAK silencing by lentiviral short hairpin RNA (shRNA) induced expression and phosphorylation of p53 at Ser15. However, FAK regulation of mesothelioma proliferation was not restricted to p53-dependent pathways, as demonstrated by immunoblots after FAK knockdown in JMN1B mesothelioma cells, which have mutant/inactivated p53, compared to MESO924, MESO257, MESO296, and MESO428 cells, which have nonmutant p53. Additive effects were obtained through a coordinated attack on p53, as demonstrated by immunoblots, cell viability and cell cycle analyses, showing that FAK knockdown and MDM2 inhibition (nutlin-3) induced greater p53 expression, cell apoptosis, anti-proliferative effects and cell cycle arrest, compared to either intervention alone. These findings highlight novel therapeutic opportunities in mesothelioma.

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 5031.
Abstract 5035: The MEK inhibitor AZD6244 is active in GNAQ mutant ocular melanoma cells
Authors: Ambrosini Grazia , Schwartz Gary K.
Published: February 2011

Uveal melanoma represents the most common intraocular malignancy in the United States. However, there are no effective treatments for this aggressive metastatic disease. While activating mutations in B-RAF are rare in ocular melanoma, oncogenic G protein alpha subunit q (GNAQ) mutations at codon 209 have been described in about 50% of uveal melanomas. Knock-down of GNAQ by siRNA inhibits p-ERK and the MEK inhibitor U0126 has been shown to inhibit growth of a GNAQ mutant cell line (Van Raamsdonk CD et al, Nature 2009). AZD6244 (ARRY-142886) is a MEK inhibitor now in clinical trials. Published phase I results have indicated clinical benefit in a patient with ocular melanoma (Adjei AA et al, J Clin Oncol. 2008). In view of this, we elected to test AZD6244 across a panel of ocular melanoma cell lines that differ in their GNAQ mutational status. Treatment with AZD4244 inhibited cell proliferation in a time- and dose-dependent manner, with IC50s in the 100-150nM range. This corresponded to the inhibition ERK phosphorylation and down-regulation of cyclin D1 in GNAQ mutant (both Q209L and P209L), but not in GNAQ wild-type cells. Over-expression of mutant GNAQ(Q209L) in wild type OCM290 cells, which are highly resistant to AZD6244, resulted in drug sensitization with inhibition of ERK phosphorylation. Depletion of GNAQ by siRNA in the GNAQ mutant cells resulted in inhibition of ERK phosphorylation and growth suppression. Interestingly, GNAQ depletion had no effect on ocular melanoma cells with wild type GNAQ or mutant B-RAF. We conclude that the GNAQ activating mutation in ocular melanoma can mediate sensitivity to AZD6244. This data provide a rationale for the clinical evaluation of AZD6244 in uveal melanoma.

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 5035.
Abstract 5065: Phosphorylation of Peptidyl prolyl isomerase 1 by Mixed Lineage Kinase 3 promotes breast cancer pathogenesis
Authors: Mishra Rajakishore , Sondarva Gautam , Kanthasamy Anumantha , Rana Basabi , Rana Ajay
Published: February 2011

MLK3 is a member of MLK sub-family that belongs to the family of MAP kinase kinase kinase (MAP3K). The MLK family members are characterized by the presence of signature sequences of Ser/Thr and Tyr kinases within their catalytic domain. The detailed physiological function of MLK3 is yet to be deciphered fully, however the available data suggest that in neuronal cells, MLK3 plays role of a pro-apoptotic kinase. The function of MLK3 in any type of cancer is not known. Interestingly the C-terminal tail of MLK3 contains consensus sequences for various protein-protein interactions and phosphorylation sites for numerous kinases. The C-terminal tail also contains Ser/Thr-Pro sequence, a consensus sequence for prolyl isomerase (Pin1), which has been reported to be over-expressed in cancer cells, including in breast cancer cells and primary tumors.

Our initial hypothesis was that probably Pin1 binds to MLK3 and regulates MLK3's kinase activity. Our in vivo binding indeed suggested that Pin1 binds to MLK3 via PPI domain robustly and to certain extent through WW domain. The binding of Pin1 did not alter the kinase activity of MLK3. Interestingly, counterintuitive to our initial hypothesis, we observed that MLK3 was able to phosphorylate Pin1 and alter Pin1's isomerase activity. We also identified S138 as a phosphorylation site on Pin1 and raised p-S138 antibody. Since, Pin1 is known to drive G2-M-cell cycle transition, we examined whether phosphorylation of Pin1 promotes cell cycle progression. Our data indicated that phospho-S138 Pin1 increased the G2-M transition of cell cycle. Pin1 is reported to be a nuclear protein; however it is not known whether this translocation of Pin1is regulated by any protein. We observed that Pin1 was translocated to nucleus upon phosphorylation by MLK3 at S138 site and the phospho-S138 Pin1 was highly expressed in breast cancer cell lines and tumors, compared to non-transformed breast epithelial cell line and normal breast tissues respectively. The breast cancer tissue arrays analyzed also showed that phospho-S138 was highly expressed in tumors compared to normal breast tissue and the intensity of staining was significantly higher in tumors as well.

Taken together, our data suggest that phosphorylation of Pin1 by MLK3 drives G2-M transition and promote breast cancer pathogenesis. Thus targeting MLK3 or Pin1 may prove beneficial to control breast cancer 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 5065.