bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2174: MLL3 act as a tumor suppressor gene in pancreatic cancer
Authors: Gupta Sonal , Mukherjee Radha , dal Molin Marco , Goggins Michael , Hebbar Sachin , Maitra Anirban
Published: August 2012

Myeloid/lymphoid or mixed-lineage leukemia 3 (MLL3) is a histone 3-lysine 4 methyltransferase, found frequently mutated in a variety of solid tumors, including pancreatic ductal adenocarcinoma where it is the fourth most common somatically mutated gene (excluding homozygous deletions). Despite various somatic mutations, the functional role of MLL3 has not been studied in pancreatic cancer. Upon careful compilation of all the known MLL3 somatic mutations in various solid tumors, no hotspots were identified. However, regardless of the mutation status, we found significantly diminished MLL3 expression in a panel of patient-derived pancreatic tumor cell lines, tumor xenografts and archived patient tumors. Examination of the MLL3 promoter region revealed a CpG island. Methylation of the promoter region was confirmed by methylation-specific PCR in majority of the tumor cell lines and xenografts. Treatment with DNA methyltransferase inhibitor relieved the promoter methylation causing increase in MLL3 transcript expression. Further, knockdown of MLL3 by siRNA in cell lines increased anchorage-independent growth, and enhanced the migration/invasion phenotype of cancer cells. Agilent microarray analysis of two pancreatic cancer cell lines subsequent to knockdown of residual MLL3 function by siRNA, revealed a battery of potential gene targets and pathways regulated by MLL3. The most common Gene Ontology (GO) categories for differentially expressed genes include those involved in cell cycle, cell migration, and DNA repair, underscoring the phenotypes observed with MLL3 siRNA. We indeed observed an increase in MLL3 expression upon DNA damage which was independent of p53 status. Using transgenic mouse model of pancreatic cancer, we are investigating whether loss of MLL3 function in exocrine pancreas cooperates with mutant Kras to accelerate the progression of pancreatic intraepithelial neoplasia (PanIN) to invasive neoplasia.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2174. doi:1538-7445.AM2012-2174
Abstract 2180: Identification of nuclear transcription factor Y (NF-Y) as a novel key transcriptional regulator of the tumor-suppressor gene DAPK1
Authors: Schmidt Christopher R. , Oakes Christopher C. , Boutros Michael , Plass Christoph
Published: August 2012

The death-associated protein kinase 1 (DAPK1) belongs to a family of multifunctional Ser/Thr-kinases that plays a crucial role in the mediation of cell death, autophagy and tumor suppression. Loss or reduced expression of DAPK1 occurs in the majority of patients with chronic lymphocytic leukemia (CLL) and many other non-hematological tumors commonly involving epigenetic silencing. To identify novel key DAPK1 transcriptional regulators, we conducted an siRNA screen in HEK293 cells using a custom library targeting over 60 individual genes with known or predicted DNA-binding function and general potential to modulate transcription. Candidate gene selection for siRNA-mediated knock-down was based on previous genome-wide siRNA screening experiments and in silico prediction of binding sites at the DAPK1 promoter region. As endogenous readout, DAPK1 mRNA expression was monitored by qRT-PCR.Using this strategy, we identified nuclear transcription factor Y (NF-Y) as the top positive regulator of DAPK1 that reduces its expression down to 55% of the level determined in untargeted control siRNA-treated cells. NF-Y is a well known transcriptional regulator of genes involved in cell cycle control, apoptosis and stress response. NF-Y consists of 3 subunits, NF-YA, -YB and -YC, forming a trimeric complex which is essential for specific binding of CCAAT boxes located proximal to the transcriptional start sites of genes. In addition to HEK293 cells, we were able to diminish DAPK1 mRNA expression upon siRNA-mediated NF-Y knockdown in a panel of 6 different cancer cell lines. Conversely, co-overexpression of NF-YA,-YB and -YC in HEK293 cells leads to increased expression levels of DAPK1. To investigate whether NF-Y contributes directly or indirectly to DAPK1 transcriptional regulation, DAPK1 luciferase reporter constructs harboring the predicted NF-Y binding site were generated. Point mutation of the reverse CCAAT-box in the DAPK1 promoter constructs significantly reduces the promoter activity down to 60% of the wild-type promoter. Furthermore, NF-Y knockdown significantly reduces the activity of the wild-type DAPK1 luciferase construct down to 55% in reporter assays. Strikingly, the CCAAT-box mutated constructs do not show altered activity after NF-Y depletion, suggesting a direct regulation of DAPK1 by NF-Y. Taken together, these results strongly indicate a direct role for NF-Y in the regulation of basal DAPK1 transcription levels, and also propose a novel mechanism for aberrant transcriptional silencing of DAPK1 in cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2180. doi:1538-7445.AM2012-2180
Abstract 2181: Characterization of PHF11, a candidate tumor suppressor gene, in esophageal squamous cell carcinoma
Authors: Cheung Wai Ying , Ko Josephine Mun Yee , Yu Zhuoyou , Lung Maria Li
Published: August 2012

Background and aims: Esophageal squamous cell carcinoma (ESCC) has a high incidence in China, with five-year survival rates in Hong Kong of only 10.7%. Loss of chromosome 13q regions is a frequent event in ESCC. A candidate tumor suppressor gene (TSG), PHF11, located at 13q14.2 was identified from our previous chromosome transfer study and shows 100% down-regulation in 18 cancer cell lines, including 16 ESCC cell lines, compared to expression in an immortalized normal esophageal epithelial cell line. This study aims to investigate the functional role of PHF11 in ESCC and to investigate the possible role of PHF11 in transcriptional regulation or histone modification as the proteins in the plant homeodomain finger (PHF) protein family are reported to contribute to the two implicated functions. Methods: Real-time PCR was carried out to validate the expression of PHF11 in tumor tissues of ESCC patients compared to their respective normal counterparts. The two isoforms of PHF11, PHF11a and PHF11b, were cloned into expression vectors and transfected into ESCC cell lines, KYSE150 and KYSE180, respectively. Clones expressing PHF11 were established for colony formation assay and MTT assay. To identify the subcellular localization of PHF11, subcellular fractionation was performed using immortalized normal esophageal epithelial cell lines followed by SDS-PAGE and Western blotting. Results: Real-time PCR verified that 42% of the 41 patient samples showed at least 2-fold down-regulation in PHF11 expression. The colony formation assay showed that the two isoforms of PHF11, PHF11a and PHF11b, have the ability to suppress cell growth using two ESCC cell lines, KYSE150 and KYSE180. The MTT assay also showed that both isoforms of PHF11 have the ability to suppress cell proliferation. Our data showed that PHF11 is localized in the nucleus of the immortalized normal esophageal epithelial cell lines. Conclusions: It was observed that almost half of the patient samples screened showed a significant down-regulation of PHF11, suggesting its possible role as a candidate TSG. Suppression of colony growth and cell proliferation in the PHF11 expressing clones indicate that the protein may suppress tumorigenicity of ESCC cells. The nuclear localization of PHF11 observed in the immortalized normal esophageal epithelial cell line is consistent with a possible role in transcriptional regulation or histone modification, though further studies are required to further investigate this.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2181. doi:1538-7445.AM2012-2181
Abstract 2187: Plk2 loss and mTOR signaling in advanced colorectal cancer
Authors: Matthew Elizabeth M. , Yang Zhaohai , El-Deiry Wafik S.
Published: August 2012

Background: Dysregulated cell growth and proliferation can promote tumorigenesis. Mammalian target of rapamycin (mTOR), a central promotor of cell growth and proliferation, is inhibited by Tuberous Sclerosis Complex 1 (TSC1) protein. Polo-like kinases (Plk) 1 & 2 are downstream molecules in the p53 pathway. TSC1 is known to interact with Plk1 in a cell cycle-regulated, phosphorylation-dependent manner. Recently we found that Plk2 impacts mTOR signaling in the same manner as TSC1, and human lung tumor cells deficient in Plk2 grew significantly larger than control tumor cells. We hypothesized that Plk2 functions as a tumor suppressor protein and seek to investigate Plk2 function as it relates to mTOR signaling and tumor suppression. Design: This is a pilot study. Archived pathology specimens from 6 advanced colorectal adenocarcinomas (stages III & IV) were retrieved, and immunohistochemical staining for phosphorylated mTOR (serine 2448), phosphorylated ribosomal S6 (Serine 235/236), Plk2, p53, Ki67, and glucose transporter 1 (Glut1) were performed. The staining pattern was analyzed. Results: Plk2 is expressed in normal colon epithelium, with a punctate staining pattern in the supranuclear region. In colorectal adenocarcinoma, Plk2 demonstrates partial loss of expression, complete loss of expression, or disrupted expression manifested as irregular or abnormal localization. Loss of Plk2 expression is more pronounced in the invasive front in some cases. mTOR shows strong expression in the invasive front, which is presumed to be less hypoxic. Phosphorylated S6 and Glut1 expression partially correlate with phosphorylated mTOR expression but appears more diffuse in some cases. p53 and Ki67 expression is diffuse, rather than specific to the tumor invasive front in the subset of cases examined. Conclusion: Our results support the hypothesis that Plk2 functions as a tumor suppressor molecule. Loss of Plk2 expression along with accentuated expression of phosphorylated mTOR, phosphorylated S6, and Glut1 at the invasive front at least in some advanced colorectal carcinomas suggest that interaction between Plk2 and TSC1 / mTOR signaling molecules plays a role in tumor progression during hypoxic condition. Further study with a larger number of tumor samples and tumor cell lines is needed to confirm the above findings.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2187. doi:1538-7445.AM2012-2187
Abstract 219: Glycosylation-dependent effect of CD109 on TGF-beta1 and EGF signaling in human glioblastoma cells
Authors: Murakumo Yoshiki , Takahashi Masahide
Published: August 2012

CD109, a glycosylphosphatidylinositol-anchored cell surface glycoprotein, is a negative regulator of TGF-beta1 signaling, whose expression is predominantly detected in squamous cell carcinomas of esophagus, lung, uterus and oral cavity. In the Northern blot analysis using various human tumor cell lines, we found CD109 expression in cell lines derived from human glioblastomas, however, the significance of CD109 in glioblastoma cells has not yet been elucidated. Here, we present the glycosylation-dependent effect of CD109 on TGF-beta1 and EGF signaling in glioblastoma cells. SK-MG-1 cells overexpressing CD109 exhibited downregulation of Smad2 phosphorylation after TGF-beta1 stimulation and upregulation of EGFR and ERK1/2 phosphorylation after EGF stimulation, indicating that CD109 is involved in not only negative regulation of TGF-beta1 signaling but also positive regulation of EGF signaling. However, in other glioblastoma cell lines, U251MG and MG178, CD109 overexpression showed no significant effect on TGF-beta1 and EGF signaling. CD109 overexpression also enhanced cell proliferation and migration in SK-MG-1 cells, but not in the other two cells. Western blot analysis showed that molecular mass of CD109 of SK-MG-1 cells was a bit high compared with those of the other two cell lines, and deglycosylation analysis revealed enhanced glycosylation of CD109 in SK-MG-1 cells. TGF-beta1 signal in U251MG and MG178 was suppressed when the medium with hyperglycosylated soluble form of CD109 was used for TGF-beta1 stimulation. Our results suggest the glycosylation-dependent effect of CD109 on cell biology of glioblastoma cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 219. doi:1538-7445.AM2012-219
Abstract 2191: Mutational analysis of DNMT3A gene in acute leukemias and common solid cancers
Authors: Yoo Nam Jin , Kim Min Sung , Lee Sug Hyung
Published: August 2012

DNMT3A, a DNA methyltransferase that functions for de novo methylation, is important in development and many cellular processes related to tumorigenesis. Somatic mutations of DNMT3A gene, including recurrent mutations in its Arg-882, were recently reported in acute myelogenous leukemia (AML), strongly suggesting its role in development of AML. To see whether DNMT3A mutation occurs in other malignancies as well, we analyzed DNMT3A in 916 cancer tissues from 401 hematlogoic malignancies (AML, acute lymphoblastic leukemias (ALL), multiple myelomas and lymphomas) and 515 carcinomas (lung, breast, prostate, colorectal and gastric carcinomas) by a single-strand conformation polymorphism (SSCP) assay. We identified DNMT3A mutations, especially the Arg-882 mutations, in adulthood AML (9.4%). In addition, we found DNMT3A mutations in a pre-B-ALL and three lung cancers at lower frequencies. Allelic loss of DNMT3A was frequently observed in most cancer types analyzed, including lymphomas (48.1%), gastric cancers (23.5%) and lung cancers (18.3%) irrespective of DNMT3A mutation. Also, loss of DNMT3A expression was common in lung cancers (46.4%) and was associated with the allelic loss. Our data indicate that DNMT3A gene is mutated mainly in AML but it occurs in other cancers such as ALL and lung cancer despite the lower incidences. Also, the data suggest that DNMT3A is altered in many cancers types by various ways, including somatic mutations, allelic loss and loss of expression that might play roles in tumorigenesis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2191. doi:1538-7445.AM2012-2191
Abstract 2196: Upregulation of survivin during immortalization is linked to repression of p16INK4a and confers resistance to oxidative stress
Authors: Petti Carlotta , Kan Chin-Yi , Dietrich Philipp A. , Lauryn Bracken , Xu Ning , Yuan Jun , Liu Tao , MacKenzie Karen L.
Published: August 2012

Survivin is an essential component of the chromosomal passenger complex and a member of the Inhibitor of Apoptosis (IAP) family. It is expressed at high levels in a large variety of malignancies, where it is implicated in cytoprotection and drug resistance. Nevertheless, the pathways that mediate the cytoprotective functions of survivin and the mechanisms underlying its upregulation in cancer have not been fully elucidated. Results from our past investigations revealed that survivin was upregulated during telomerase-mediated immortalization of human myofibroblasts (MRC-5) (1). Since the immortalization process is an essential step in human carcinogenesis, upregulation of survivin during immortalization may explain why survivin is abundant in virtually all cancers. The present study aimed to characterize the mechanism responsible for the upregulation of survivin during immortalization, and the functional consequences of its over expression in immortal cells. Results from whole genome expression analyses and qRT-PCR demonstrated that the upregulation of survivin during immortalization was paralleled by increased expression of the transcription factor E2F1. The chromatin immunoprecipitation (ChIP) assay confirmed that E2F1 directly interacted with the survivin gene (BIRC5) promoter in immortal cells. Moreover, it was shown that E2F1 binding to BIRC5 was corresponded with repression of tumor suppressor p16INK4a, an event that commonly occurs during immortalization and human tumorigenesis. Survivin and p16INK4a were also functionally linked by assays that showed either upregulation of survivin or repression of p16INK4a rendered telomerase-transduced MRC-5 cells resistant to oxidative stress. Conversely, siRNA-mediated down regulation of survivin activated caspases and enhanced the sensitivity of immortal MRC-5 cells to oxidative stress. Together these data show that repression of p16INK4a contributes to the upregulation of survivin, and thereby provides a survival advantage to cells that are subject to oxidative stress during immortalization. 1. Yuan et al., Oncogene (2009) 28, 2678-2689.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2196. doi:1538-7445.AM2012-2196
Abstract 220: TNFα activates NF-κB through RIP1 ubiquitination-dependent and independent pathways
Authors: Zhang Laiqun , Blackwell Ken , Workman Lauren , Habelhah Hasem
Published: August 2012

TNFα-induced NF-κB activation has long been believed to depend on TRAF2/5- and cIAP1/2-mediated RIP1 ubiquitination. Recently, HOIP/HOIL-1-mediated linear ubiquitination of NEMO/IKKγ has emerged as a new player in TNFα-induced NF-κB activation. However, the mechanisms by which these proteins mediate NF-κB activation have become more complex than ever before due to an accumulation of conflicting results. Several independent studies even challenged the notion that these proteins ever play essential roles in NF-κB activation. Here, we report the identification of immediate and delayed phases of IKK activation in TNFα-treated cells. TNFα-induced immediate IKK activation requires ubiquitination of RIP1 catalyzed by cooperative actions of TRAF2, cIAP1/2, UbcH5 and Ubc13, and that in the absence of any one of these components; those remaining are only capable of mediating delayed IKK activation by recruiting the HOIP/HOIL-1 complex. In RIP1-deficient cells, TRAF2 undergoes increased auto-ubiquitination through K63-linkage, yet it is not capable of mediating immediate IKK activation. Notably, depletion of either HOIP in RIP1-deficient cells or both TRAF2 and cIAP1/2 in wild-type cells completely impairs IKK activation. In addition, deletion of the TRAF2 RING domain also severely reduces the immediate phase of IKK activation. These data suggest that two RING finger E3 ligases (TRAF2 and cIAP1) and two E2 enzymes (UbcH5 and Ubc13) cooperate to catalyze immediate RIP1 ubiquitination and IKK activation, and that, in the absence of RIP1, TRAF2 and cIAP1/2 recruit HOIP/HOIL-1 and mediate the delayed phase of IKK activation. TRAF5 has no substantial role in TNFα-induced NF-κB activation. Importantly, in embryonic hepatocytes, RIP1 is completely degraded, yet these cells are not sensitized to TNFα-triggered cell death because of delayed IKK activation. Thus, this study provides evidences for the existence of RIP1 ubiquitination-dependent and -independent pathways that mediate NF-κB activation, and thereby clarifies molecular details of this important signaling mechanism.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 220. doi:1538-7445.AM2012-220
Abstract 2200: Identification of GLI1 binding partners that regulate transactivation or cell transformation
Authors: Yoon Joon Won , Higashiyama Nicole , Lamm Marilyn , Iannaccone Philip M. , Walterhouse David O.
Published: August 2012

Activation of the GLI1 transcription factor and oncogene appears to play a role in the genesis of desmoplastic medulloblastomas, basal cell carcinomas, and some rhabdomyosarcomas. We identified interactions between GLI1 and proteins that enhance the transactivating or cell transforming function of GLI1. Such protein-protein interactions could potentially be targeted in the treatment of some GLI1-related malignancies. We focused on proteins interacting with GLI1 transactivation domains, believing these would be essential for transactivation and cell transformation. Previously, we identified an acidic alpha-helical transcription activation domain (TAD1; aa 1020-1091) at the carboxyl terminus of the GLI1 protein. We show that TAD1 physically interacts with the coactivator TAF9 in vitro by pull down assays and in rhabdomyosarcoma cells in vivo by proximity ligation assays and co-immunoprecipitation. Inhibition of this interaction reduces transactivation by GLI1 and the ability of GLI1 to transform RK3E cells. We identify a second GLI1 transactivation domain (TAD2; aa 635-740), which activates reporter gene transcription and accounts for the TAD1-independent cell transforming ability of GLI1. TAD2 contains conserved sequences among GLI1 family proteins, including a CBP binding domain identified in GLI3. However, consistent with in vitro data from others, we could not demonstrate interaction of TAD2 and CBP in rhabdomyosarcoma cells in vivo by proximity ligation assay. To identify TAD2 binding proteins, we performed yeast 2 hybrid screening and identified other transcription factors (Y-Box1, HSF2, RBMX2, and ZMYM2), cytoskeleton-associated proteins and membrane-associated proteins. Based on a previously described GLI1-p53 inhibitory loop that controls stem cell and tumor cell numbers, we demonstrate that p53 down-regulates TAD1 but not TAD2 function. Understanding the mechanism of transactivation and cell transformation by GLI1 can provide novel targeted approaches to inhibit the GLI1 transcription factor in cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2200. doi:1538-7445.AM2012-2200
Abstract 2203: AP-1 activation and a functional SNP242 regulates the LY-6K gene expression in human breast cancer cells
Authors: Kong Hyun Kyung , Park Jong Hoon
Published: August 2012

Background: Lymphocyte antigen 6 complex locus K(LY-6K), a member of LY-6/uPAR receptor superfamily and cancer-testis antigen, is a novel breast cancer biomarker. Also LY-6K is target antigen for the head and neck squamous cell carcinoma, and lung and esophageal carcinoma. Elevated LY-6K induces cell invasion and metastasis through activating the Raf-1/MEK/ERK signaling pathway. However, the molecular mechanisms that mediate regulation of human LY-6K gene expression are completely unknown. Material and Methods: Within the pGL3-basic reporter vector, the −3050/+182 bp fragment, truncated and mutated fragment upstream of the LY-6K gene was subcloned and measured promoter activity. Subsequently, multiple cis elements were identified using several gene tool software (TFSEARCH, TRANSFAC, PROMO and MATCHTM) in the promoter of human LY-6K gene. We investigated the transcription factor and the effect of single nucleotide polymorphism (SNP) on LY-6K promoter activity and transcription factor binding using chromatin immunoprecipitation (ChIP) assays, electrophoretic mobility shift assays (EMSA), and supershift assays. Results: LY-6K gene was expressed in estrogen receptor (ERα) and progesterone receptor (PR)-negative breast cancer cell lines. A novel AP-1 binding site in the LY-6K promoter regulates LY-6K gene expression. A polymorphic C/G-SNP at the 242 nucleotide position in the promoter region (rs2585175, called SNP242) was validated to be located in a PAX-3 transcription binding motif, which is appear with the presence of the G-allele resulting in a ∼54% decrease in LY-6K promoter activity by interfering binding of AP-1 transcription factor. Discussion: The activity of the AP-1 transcription factor plays an important role in cell mobility of breast cancer cells via the promoting of LY-6K gene expression. LY-6K expression is dependent on not AP-1 transcription factor, but also functional SNP242 G-allele. These results suggested that the polymorphism rs2585175 (SNP242), in close proximity of the LY-6K promoter, lead to LY-6K downregulation, and thus, may reduce the risk of breast cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2203. doi:1538-7445.AM2012-2203
Abstract 2204: BCL11B is a critical EWS/FLI up-regulated target in Ewing's sarcoma
Authors: Toomey Elizabeth , Lui Bianca , Lessnick Stephen
Published: August 2012

Objective: Ewing's sarcoma is a highly aggressive tumor of the bone and soft tissue that affects the pediatric and young adult population, and is characterized by the EWS/FLI chromosomal translocation. This fusion protein acts as an oncogenic transcription factor to dysregulate the expression of a number of genes that contribute to the transformed phenotype. One challenge is to define which of these dysregulated genes are important for the oncogenic phenotype of Ewing's sarcoma. To address this issue, we used a proteomics approach to identify new protein-protein interactions among target genes of EWS/FLI. This screen identified BCL11B, which had previously been identified in genome-wide transcriptional profiling studies by our group, and others, as an up-regulated target of EWS/FLI. Methods: To define the function of BCL11B in Ewing's sarcoma, we used retroviral mediated shRNA knock-down of EWS/FLI, followed by re-expression of the EWS/FLI cDNA, to verify that BCL11B expression is modulated by the fusion protein. Anchorage independent growth assays assessed the oncogenic potential of patient derived Ewing's sarcoma cell lines in which BCL11B had been knocked-down with shRNA. Luciferase assays and chromatin immunoprecipitation techniques were used to interrogate BCL11B regulation by EWS/FLI. Results: Our results show that BCL11B is up-regulated by EWS/FLI and is necessary for oncogenic transformation in patient derived Ewing's sarcoma cell lines. Our work addressing the mechanism of BCL11B regulation by EWS/FLI is on-going, but preliminary data suggests that BCL11B may be a direct target of EWS/FLI. Conclusion: This work identified BCL11B as an EWS/FLI up-regulated target that contributes to transformation in Ewing's sarcoma. It also provides a setting in which we can broaden our knowledge regarding the mechanisms used by EWS/FLI to up-regulate critical target genes.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2204. doi:1538-7445.AM2012-2204
Abstract 2207: SUMOylation of EAPII/TTRAP/TDP2 modulates its function
Authors: Li Chunyang , Khuri Fadlo R. , Sun Shi-yong , Li Runzhao
Published: August 2012

ETS1 Associated Protein II (EAPII, also called TTRAP, TDP2) was originally identified by two-hybrid screening as a novel protein that interacts with both ETS1 and the cytoplasmic domain of CD40 receptor. EAPII may represent a novel component in signal transduction processes that regulates eukaryotic gene expression in response to extracellular signals. In support of this hypothesis, we have demonstrated that EAPII negatively modulates ETS1 transcriptional activity and attenuates synergistic transactivation by ETS1 and NFκB. However, the mechanism by which EAPII transduces signals is elusive. In this report, we provide the evidence of EAPII protein sumoylation in vivo. Sumoylation is an important posttranslational modification that involves conjugation of the small ubiquitin-like modifier (SUMO) to lysine residues of the targeting proteins. EAPII is sumoylated by both SUMO-1 and SUMO-2/3. Sumoylation analysis of cell fractions revealed that sumoylation by SUMO-2/3 mainly occurs in the nucleus while that by SUMO-1 in cytoplasm. Since EAPII protein possesses no SUMO consensus motif (≥KxE), we constructed a series of EAPII truncated mutations to determine the potential sumoylation site(s). Our data indicated that the noconsensus sumoylation site(s) may reside between amino acid residues 68 and 279. Interestingly, C-terminal deletion (Del 279-362) significantly increases the sumoylation, suggesting that the C-terminus might contain an inhibitory domain for EAPII sumoylation. It has been hypothesized that sumoylation may mediate extracellular signals in response to stresses, hypoxia and cytokines. Whether EAPII sumoylation interferes with its repressive activity on ETS1 transcription remains to be elucidated, and determination of the signaling pathways that may modulate EAPII sumoylation will provide a better understanding of the modulation of ETS1 functions in specific pathphysiological microenvironments.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2207. doi:1538-7445.AM2012-2207
Abstract 2211: Accessible chromatin structure permits factors Sp1 and Sp3 to regulate human TGFBI gene expression
Authors: Lee Jong-Joo , Park Joo-Hong , Park Keunhee , Song Min-Ji , Yang Wook-Jin , Kim Hyoung-Pyo
Published: August 2012

Transforming growth factor beta 1-induced (TGFBI) protein is an extracellular matrix (ECM) protein expressed widely in human tissues. The gene encoding this protein was first described as a major TGF-b-responsive gene in the lung adenocarcinoma cell line A549. TGFBI appears to mediate cell adhesion and migration through interaction with integrin, and increased TGFBI expression has been associated with inflammatory diseases including rheumatoid arthritis, atherosclerosis, cyclosporine nephropathy, and stab wound healing. TGFBI is ubiquitously expressed in healthy tissues, while disrupted TGFBI expression has been described in human tumor cell lines and in human primary tumor samples. A general function of this protein appears to be as a promoter or suppressor of cancer progression, depending on the tissue. Regulation of TGFBI expression by epigenetic mechanism such as DNA methylation and microRNA has been reported, but few studies have focused on how chromatin structure affects transcriptional activity relating to TGFBI expression. We show that both chromatin structure and regulatory elements in the TGFBI promoter region that bind factors Sp1 and Sp3 regulate constitutive expression of TGFBI. In this study, we investigated how human TGFBI expression is regulated in lung and breast cancer cells. We observed that the TGFBI promoter in A549 and MBA-MD-231 cells, which constitutively express TGFBI, existed in an open chromatin conformation associated with transcriptionally permissive histone modifications. Moreover, we found that TGFBI expression required Sp1 transcription elements that can bind transcription factors Sp1 and Sp3 in vitro. Occupancy of the TGFBI promoter by Sp1 and Sp3 in vivo was only observed in TGFBI-expressing cells, indicating that open chromatin conformation might facilitate the binding of Sp1 and Sp3 to the TGFBI promoter region. TGFBI promoter activity was impaired when Sp1 elements were mutated, but was increased when Sp1 or Sp3 factors was overexpressed. Furthermore, Sp1 inhibition in vivo by mithramycin A, as well as knockdown of Sp1 and/or Sp3 expression by short interfering RNA, significantly reduced TGFBI mRNA and protein levels. Thus, our data demonstrated that the expression of TGFBI is well correlated with chromatin conformation at the TGFBI promoter, and that factors Sp1 and Sp3 are the primary determinants for the control of constitutive expression of TGFBI gene.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2211. doi:1538-7445.AM2012-2211
Abstract 222: NEK6 promotes JB6 C141 cell transformation and activates STAT3 through its phosphorylation on Ser727
Authors: Lee Kun Yeong , Jeon Young Jin , Bode Ann M. , Dong Zigang
Published: August 2012

NEK6 (NIMA-related kinase 6) is a homologue of the Aspergillus nidulans protein NIMA (never in mitosis, gene A). We demonstrate that ectopic expression of NEK6 induces anchorage-independent transformation of JB6 Cl41 mouse epidermal cells. Tissue arrays and Western immunoblot analysis show that NEK6 is overexpressed in malignant tissues and several cancer cell lines. We also discovered that NEK6 interacts with STAT3, an oncogenic transcription factor, and phosphorylates STAT3 on Ser(727), which is important for transcriptional activation. Additional studies using NEK6 mutants suggested that the phosphorylation on both Ser(206) and Thr(210) of NEK6 is critical for STAT3 phosphorylation and anchorage-independent transformation of mouse epidermal cells. Notably, knockdown of NEK6 decreased colony formation and STAT3 Ser(727) phosphorylation. Based on our findings, the most likely mechanism that can account for this biological effect involves the activation of STAT3 through the phosphorylation on Ser(727). Because of the critical role that STAT3 plays in mediating oncogenesis, the stimulatory effects of NEK6 on STAT3 and cell transformation suggest that this family of serine/threonine kinases might represent a novel chemotherapeutic target.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 222. doi:1538-7445.AM2012-222
Abstract 2224: Targeted PI3K/mTOR inhibition impairs tumor cell motility and bone metastatic outgrowth via modulation of p27
Authors: Besser Alexandra H. , Wander Seth A. , Zhao Dekuang , Hong Feng , Wei Jian Qin , Wang Bin , Ince Tan , Milikowski Clara , Nadji Mehrdad , Briegel Karoline , Bishopric Nanette , Slingerland Joyce M.
Published: August 2012

The pleiotropic oncogenic effects of PI3K/mTOR have stimulated development of catalytic PI3K and mTOR inhibitors for the treatment of cancer. Present data provide a novel rationale for the use of dual PI3K/mTOR inhibitors to oppose tumor motility, invasion and metastasis. Cytoplasmic mislocalization of p27 is implicated as a key downstream driver of PI3K/mTOR-dependent tumor cell motility in vitro and bone metastatic outgrowth in vivo. Here, we report that PI3K/mTOR-kinase inhibition with PF-04691502 impairs breast cancer metastasis to bone, in part via effects on p27. Low-dose PF-04691502 impaired motility and invasion of highly PI3K/mTOR activated, bone-metastatic MDA-MB-1833 cells in vitro without affecting apoptosis or proliferation. Moreover, drug pre-treatment impaired bone metastasis in vivo. PF-04691502 decreased cytoplasmic p27pT157 and p27pT198, and p27T157D/T198D overexpression conferred resistance to inhibition of motility by PF-04691502. p27 knockdown in MDA-MB-1833 relieved RhoA-ROCK inhibition, decreased cell motility/invasion in vitro, and bone metastasis in vivo. p27 knockdown also reversed EMT phenotypic markers in MDA-MB-1833, as did PI3K/mTOR inhibition, demonstrating that p27 may be critical for maintenance of the EMT cellular program. Indeed, loss of cytoplasmic p27 reversed expression of an EMT gene profile supporting the notion that p27 acts as a key mediator of metastasis downstream of PI3K/mTOR. Cytoplasmic p27 in human cancers may prove to be a useful predictive marker to assess both PI3K/mTOR activation and the potential efficacy of catalytic PI3K/mTOR inhibitors.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2224. doi:1538-7445.AM2012-2224
Abstract 2225: GSK3β plays a significant role in Ras driven tumors potentially through an upstream effect on AKT
Authors: Sweeting Raeshell S. , Kim Hong J. , Baldwin Albert S.
Published: August 2012

Pancreatic cancer is a highly aggressive cancer that is resistant to most drug therapies. Lung cancer is the number one cause of cancer deaths in the US and worldwide. Both of these tumors are known to have high levels of Ras oncogenic mutations (50 and 90% respectively). AKT is known to phosphorylate and inactive Glycogen Synthase Kinase 3 (GSK3). Additionally, GSK3 has been shown to play a role in oncogenesis in pancreatic cancer and lung cancer. Given the high level of AKT activity in transformed cells these two processes would appear to be at odds. However, much of the work on GSK3 has focused on the beta subunit. We have found that the alpha subunit plays a role in tumor proliferation and AKT activity. We see a significant effect of knockdown of GSK3β in Ras transformed pancreatic (Panc1, MiaPaca) and lung cancer (A549) cell lines in proliferation assays, with an approximately 70% decrease in growth compared to siControl cells. In contrast, the knockdown of the GSK3β subunit resulted in no decrease in cell proliferation in A549 cells compared to a 45% decrease in Panc 1 cells. Additionally, knockdown of GSK3β resulted in decreased levels of AKT activity by Western blot analysis as compared to siControl and GSK3β. Previous studies have focused on the role of the beta subunit of GSK3 in oncogenesis. Additionally, AKT has been studied as an upstream regulator rather than a downsteam target of GSK3. Our early studies suggest that the GSK3β subunit may play a significant role in the growth of Ras driven tumors cell lines upstream of AKT, and should be a potential target for therapeutic strategies.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2225. doi:1538-7445.AM2012-2225
Abstract 2232: Overexpression of the mu opioid receptor in human non-small cell lung cancer promotes Akt and mTOR activation, tumor growth and metastasis
Authors: Lennnon Frances E. , Mirzapoiazova Tamara , Mambetsariev Bolot , Salgia Ravi , Moss Jonathon , Singleton Patrick A.
Published: August 2012

Background: Recent epidemiologic studies suggesting that there were differences in cancer recurrence contingent on anesthetic regimens have raised the possibility that mu opioid agonists can influence cancer progression. Based on our previous studies indicating the mu opioid receptor (MOR) is upregulated in several types of non-small cell lung cancer (NSCLC), this study examined the functional significance of MOR overexpression to elucidate a possible mechanism for the epidemiologic findings. Methods: Stable vector control (VC) and MOR1 overexpressing human NSCLC bronchioloalveolar carcinoma (BAC) cells were evaluated using immunoblot analysis, proliferation and transendothelial extravasation assays with or without Akt inhibitor, mTOR inhibitor (temsirolimus) or the peripheral MOR antagonist, methylnaltrexone (MNTX). In human NSCLC xenograft models, primary tumor growth rates and lung metastasis were analyzed using consecutive tumor volume measurements and nestin immunoreactivity in nude mouse lungs. Results: We provide evidence that MOR is an important regulator of lung cancer progression, a disease with high mortality and few treatment options. MOR overexpression increased Akt and mTOR activation, proliferation and extravasation in human BAC cells. In vivo, overexpression of MOR in BAC increased primary tumor growth rates in nude mice by ∼2.5 fold and lung metastasis by ∼20 fold compared to VC BAC cells. Conclusions: Our overexpression data suggests a possible direct effect of MOR on Akt and mTOR activation and lung cancer progression. Such an effect provides a plausible explanation for the epidemiologic findings. Our observations further suggest that exploration of MOR in NSCLC merits further study both as a diagnostic and therapeutic option.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2232. doi:1538-7445.AM2012-2232
Abstract 2234: Activation of PI3K pathway by P-Rex2 is inhibited by GNMT
Authors: Li Chung-Hsien , Yen Chia-Hung , Chen Mei-Yu , Chen Yi-Ming
Published: August 2012

Glycine N-methyltransferase (GNMT) is a tumor suppressor for hepatocellular carcinoma (HCC). Previously, we reported that GNMT regulates mTOR signaling pathway through interacting with an mTOR binding protein-DEPTOR. Sequence blasting and phylogenetic analysis demonstrated that the first DEP domain of DEPTOR clustered with P-Rex2, a regulator of the small guanosine triphosphatase Rac. Since P-Rex2 has been showed to be able to antagonize PTEN in PI3K pathway, the aims of this study was to analyze the interaction between GNMT and P-Rex2 and their association in PI3K-Akt signaling pathway. Gel filtration assay demonstrated that GNMT and P-Rex2 were eluted in the same fractions. Reciprocal co-immunoprecipitation experiments demonstrated that GNMT interacts with different domains of P-Rex2 including DH, PH, PDZ and InsPx4-Phosphatase. Overexpression of P-Rex2 enhanced Akt activation in HCC cell line as well as in breast cancer cell lines and such effect was blocked by over-expression of GNMT. Pulse-chase experiments showed that overexpression of GNMT caused significant reduction of the half-life of P-Rex2 from 15 hr to 9.5 hr. Finally, overexpression of GNMT in HuH-7 cells resulted in a proteasome-dependent degradation of P-Rex2. Therefore, GNMT affects PI3K-Akt pathway via direct binding with P-Rex2 and enhance its degradation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2234. doi:1538-7445.AM2012-2234
Abstract 2243: Targeting the MUC1-C oncoprotein synergistically enhances cytotoxicity of breast carcinoma cells in combination with PI3K inhibitors
Authors: Raina Deepak , Kharbanda Surender , Kufe Donald
Published: August 2012

The mucin 1 (MUC1) heterodimeric protein is aberrantly overexpressed by most human breast carcinomas and contributes to the malignant phenotype. However, there are no effective agents that directly inhibit the oncogenic MUC1-C subunit and induce death of breast cancer cells. We have synthesized MUC1-C peptide inhibitors that bind to the MUC1-C cytoplasmic domain and block the formation of MUC1-C dimers in breast cancer cells. We have shown that inhibition of MUC1-C arrests growth and induces necrotic death of the breast carcinoma cells. Here we demonstrate that combining the MUC1-C inhibitor GO-203-2C with agents that target PI3K (LY294002, GDC0941) induces highly synergistic cytotoxicity of breast carcinoma cells. Notably, GO-203-2C significantly enhanced the effectiveness of these PI3K inhibitors by contributing to the downregulation of p-AKT and p-S6K. Taken together, these findings provide support for combining PI3K inhibitors and GO-203-2C, an agent now undergoing Phase I evaluation, to improve treatment of patients with breast cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2243. doi:1538-7445.AM2012-2243
Abstract 2256: PDEF regulation and role as a survival factor in ER positive breast cancer cells
Authors: Buchwalter Gilles , Brown Myles
Published: August 2012

PDEF (Prostate Derived Ets Factor) is a member of the ‘Ets’ family. These transcription factors regulate a number of biological processes, including cell proliferation, differentiation, invasion and are thought to play an important role in oncogenesis. PDEF is expressed exclusively in tissues with a high epithelial content, especially hormone-regulated epithelium, such as prostate, mammary gland, endometrium and ovary. Furthermore, several studies showed PDEF to be one of the most highly over expressed mRNA in human and mouse mammary tumors. PDEF is interestingly significantly co-expressed with ER in ER positive breast carcinomas, together with known ER cofactors such as FOXA1, XBP1 and GATA3. PDEF was previously characterized as an Androgen Receptor cooperating factor in prostate, however its potential function as an ER cofactor is not explored yet. We investigated the mechanism of this ER/PDEF co expression and the role of PDEF in breast cancer cells. Using siRNA downregulations, mRNA quantification, and Chromatin IP, we discovered that the mechanism of ER/PDEF co expression implies a critical role for GATA3 as a negative regulator of PDEF expression. Moreover PDEF and GATA3 expressions are inversely correlated in ER positive Breast Cancer and cell lines. Kaplan-Meier survival analyses showed that these two factors could be useful prognosis markers for patients. We performed growth curves, cell cycle (FACs) and apoptosis (Annexin V) analyses of MCF7 cells transfected with a PDEF targeting siRNAs. The growth curves revealed an impaired proliferation rate of PDEF downregulated cells. Quantification of apoptosis in various conditions (basal, stress induced) demonstrated that PDEF is an essential survival factor for breast cancer cells. The loss of PDEF expression indeed triggers an increased proportion of apoptotic cells, especially in stress conditions such as hormone depletion or tamoxifen / fulvestrant treatments. The findings presented in this poster are evidences that designate PDEF as a potential new cofactor of ER and as an interesting therapeutic target, since its down regulation makes breast cancer cells more prone to undergo apoptosis in stress conditions.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2256. doi:1538-7445.AM2012-2256