bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 116: A novel methodology for analyzing post-processed LC/MS metabolomics data
Authors: Mak Tytus , Tyburski John B. , Kalinich John F. , Fornace Albert J.
Published: February 2011

Metabolomics, the global study of small molecule complements, shows great promise as a tool for the discovery of biomarkers of mutagen exposure and early tumorigenesis. With the ever increasing pool of quantitative data yielded from metabolomic research, methods and tools with which to analyze and extract meaningful conclusions from these data are becoming more and more crucial. Methods currently in wide use are limited in either the scope and depth of analysis or are not specifically tailored to metabolomic datasets. In light of this, a new methodology has been developed and presented here that is specifically tailored to characteristics unique to post-processed LC/MS metabolomic data acquired from biofluid samples. For this study, twenty-four-hour urine samples were collected from male rats before and after whole body exposures to doses of gamma radiation ranging from 0.5 to 10 Gy (n = 20 per dose). Urine metabolomics data were acquired by Ultra-Performance Liquid Chromatography coupled to time-of-flight mass spectrometry, operated in both positive and negative electrospray ionization modes, and pre-processed using MarkerLynx software (Waters, Inc.). Relative abundances of urinary ions were normalized to corresponding creatinine relative abundances to reduce the probability of confounding by variation in renal function. These data were then used to develop our methodology, which can be used as a visualization tool effective in qualitatively illustrating an overall metabolome response to radiation exposure. This is achieved by creating 3D plots, on which each plotted point represents a metabolite, specified by its mass/charge (X axis) and retention time (Y axis), with its pre- to post-exposure change in regulation (Z axis) colorized with regard to the magnitude of change. In addition, numerous data filters and data augmentation schemes, such as noise filters, were developed in order to yield results that were more quantitatively robust, from which stronger conclusions can be drawn about the experiment, such as identifying specific metabolites of interest. With the development of this methodology, the repertoire of analytical tools available to metabolomics researchers will be enhanced, facilitating more rapid and meaningful biomarker discovery.

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 116.
Abstract 1160: Whole genome amplification and high-throughput sequencing of formalin-fixed paraffin-embedded colorectal cancer
Authors: Myllykangas Samuel , Buenrostro Jason , Bell John , Ji Hanlee P.
Published: February 2011

Formalin-fixed paraffin-embedded (FFPE) tumors represent a valuable resource for translational cancer genomic research. However, genetic analysis of the FFPE material is challenging, as archived tissues are often small biopsies and the fixation process damages DNA. In researching a solution to this, we have evaluated the performance of different whole genome amplification methods for obtaining DNA samples from archived tumors and their use as a template in high-throughput sequencing.

Two whole genome amplification methods were applied using 20 μm sections of FFPE colorectal cancer tissue and normal human genome DNA samples. Genomic DNA was amplified using Multiple Displacement Amplification (MDA) method and a modified version of Degenerate Oligonucleotide PCR (DOP) assay. Non-amplified human genomic DNA was used as a reference when genomic coverage, linearity and uniformity of the amplified products were assessed. Paired-end sequencing libraries were prepared from the DOP amplified templates while the MDA samples underwent single read library preparation. Sequencing was performed using an Illumina Genome Analyzer II.

2.5 million 54-mer reads mapping to the human genome were generated for each sample. Whole genome amplification was shown to produce artifacts when compared to non-amplified genomic DNA sequences. The modified DOP method preferentially amplified non-repetitive sequences in the human genome. Using the MDA-based method, genomic sequence coverage was generally more even between regions of repetitive and non-repetitive DNA. However, spread of the sequence read counts was greater in the MDA method and MDA amplification offered less dynamic range to identify copy number aberrations. Using whole genome amplification methods with the FFPE colorectal tumor sample we were able to generate 130 Mb of genomic sequence data and identify DNA copy number gains in chromosomes 13 and 20q.

Whole genome amplification and high-throughput sequencing can be used to generate sequence data from minute amounts of FFPE material and identify DNA copy number aberrations in the archived tumors. Nonetheless, whole genome amplification produces artifacts by introducing bias towards specific regions of the genome, generating artificial and random DNA fragment assemblies (MDA) and introducing universal primer sequence in the ends of the amplified DNA fragments (DOP).

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 1160.
Abstract 1168: Urothelial cell carcinomas harbor very frequent mutations in FGFR3-PI3K-AKT pathway and highest prevalence of mutations is related to low-grade tumors
Authors: Hernandez Silvia , Agell Laia , Muga Silvia De , Juanpere Nuria , Lopez Laura , Murillo Raquel , Lorenzo Marta , Serrano Sergio , Bellmunt Joaquim , Lloreta Josep
Published: February 2011

More than 90% of bladder cancers are urothelial cell carcinomas. Superficial/papillary and invasive tumors progress via different molecular pathways. FGFR3 and PI3KCA mutations are more commonly found in superficial/papillary tumors. The PI3K-AKT pathway emerges as an alternative pathogenic mechanism linked to bladder cancer.

We have analyzed the prevalence of mutations in FGFR3, PI3KCA, KRAS, BRAF and AKT1 genes in a set of 61 bladder tumors classified as TaG1 (n=10), TaG2 (n=19), TaG3 (n=8), T1G2 (n= 3), T1G3 (n= 11), T2G2 (n=1) and T2G3 (n=10). We focused on analyzing the hot spot mutation codons and surrounding regions. Mutation analysis of DNA from paraffin-embedded tumors was performed by direct sequencing of PCR products. Thirty-six (60%) tumors presented mutations, 22 cases with only one mutated gene, whereas 14 of 61 cases had mutations in two genes. The most frequently mutated gene was FGFR3 in 25 tumors, followed by PI3KCA in 16 tumors. The distribution and combinations of mutations was: FGFR3 (n= 16), PI3KCA (n= 3), AKT1 (n= 2), KRAS (n= 1), FGFR3-PI3KCA (n= 9), FGFR3-AKT1 (n= 1), and PI3KCA-KRAS (n= 4). No tumor showed BRAF mutations. According to tumor stage, 26 of 37 (70%) Ta tumors, 3 of 13 (23%) T1 tumors and 7 of 11 (63%) of T2 tumors harbored mutations. On the other hand, according to tumor grade, the distribution of mutations was: grade 1, 9 of 11 (82%); grade 2, 15 of 22 (68%); and grade 3, 12 of 28 (43%), with an overall rate of 27 mutations in 50 high grade tumors (54%). In grade 1, 100% of mutations were found in FGFR3 and/or PI3KCA, with FGFR3 mutations in 9 of 10 tumors. In grade 2, 11 of 15 mutated tumors (73%) showed alterations in FGFR3 and/or PI3KCA, and 4 tumors displayed mutations in AKT1 and KRAS (3 of them in combination with FGFR3 or PI3KCA). In grade 3, 8 of 12 mutated tumors (66%) showed mutations in FGFR3 and/or PI3KCA, and 4 tumors harbored mutations in AKT1 and KRAS (2 of them in combination with PI3KCA). Mutations in AKT1 and KRAS were found in grade 2 and 3, but not in grade 1 tumors. Percentage of mutations in the FGFR3-PI3K-AKT1 pathway is inversely proportional to grade, and it is more often associated with low grade tumors. Mutations in FGFR3 are often found alone in bladder tumors, whereas PI3KCA mutations are more frequent in combination with other mutated genes, such as FGFR3 or KRAS. FGFR3 and KRAS mutations seem to be mutually exclusive, but not PI3KCA and KRAS, nor FGFR3 and AKT1 mutations.

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 1168.
Abstract 1183: Association of germline variants of the TGFβR1 gene in colorectal cancer patients
Authors: Schaeper James P. , Murray Kevin , DeBrosse Jessica L. , Vogt Briana , Lucas Denise , Colligan Bruce M. , Deddens James A. , Douglass Larry E. , Graff Jeremy R. , Carter Julia H.
Published: February 2011

Colorectal cancer is the second leading cause of cancer death in the United States and approximately 147,000 new cases of colorectal cancer (CRC) were expected in 2009. Most people with early colorectal cancer have no symptoms and symptoms usually only appear with more advanced disease. Therefore, susceptibility markers which could possibly identify patients at risk for colorectal cancer are important for earlier detection of the disease. Germline variations in the transforming growth factor beta (TGF-β) type I receptor (TGFβR1) have been found to be associated with several human cancers, in particular with advanced ovarian cancers, suggesting a role in advanced disease stage. We analyzed two genetic variants in the TGFβR1 gene for their association with colorectal cancer. One is an intronic variant, G→A polymorphism (Int7G24A), which is 24 base pairs downstream of the intron-exon 7 boundary. The other is a nine base pair deletion (TGFβR1*6A) in exon 1 of TGFβR1. Using capillary electrophoresis, we analyzed paraffin-embedded tissue specimens from 147 patients with CRC and 169 non-cancer controls. There was no difference between male and female non-cancer controls in the prevalence of either variant. When CRC cases were compared to non-cancer controls, it was found that the prevalence of TGFβR1*6A was not significantly increased (p = 0.11) but Int7G24A prevalence was significantly increased (p = 0.000039). For CRC cases and controls, the wild type genotype was compared to patients with either or both variants and a significant difference was found primarily due to the increased prevalence of Int7G24A in CRC patients (p = 0.000052). Int7G24A was more commonly found in invasive and metastatic CRC patients as compared to non-cancer controls (p = 0.00030). We conclude that Int7G24A is a potential susceptibility marker for CRC and is associated with advanced disease stage.

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 1183.
Abstract 1192: Impedance based kinetic cellular response profiling in drug discovery and development
Authors: Ke Ning , Wang Xiaobo , Xu Xiao , Abassi Yama
Published: February 2011

A novel cell-based kinetic profiling approach using impedance readout for monitoring the effect of small molecule compounds will be described. This non-invasive readout allows continuous sampling of cellular responses to biologically active compounds and the ensuing kinetic profile provides information regarding the temporal interaction of compounds with cells. The utility of this approach was tested in two approaches. First, compounds with similar mechanism of action produced similar impedance-based time-dependent cell response profiles (TCRP). The TCRP was used to identify novel mechanisms for existing drugs and also identified potential off-target activity for COX-2 inhibitor, celecoxib and the experimental compound, monastrol. The second approach entailed using this profiling approach to screen a 120,000 compound library. We identified novel anti-mitotic compounds as well as compounds interfering with DNA synthesis. Furthermore, we also identified compounds specific for oncogene addiction pathways. We will present data in support of both approaches in this presentation.

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 1192.
Abstract 1195: Oct-1 as a potential biomarker for oxaliplatin-based therapy in non-small cell lung cancer
Authors: Kadoch Orli , Li Tong , You Liang , Yee Sook-Wah , Giacomini Kathleen , Jablons David M. , Xu Zhidong
Published: February 2011

Non-small cell lung cancer (NSCLC) remains the leading cause of cancer death in the developed world. Platinum-based chemotherapy, including agents such as cisplatin and carboplatin, represents the standard therapeutic regimen in both adjuvant and metastatic settings. However, these treatments are often associated with profound dose-limiting toxicities in some patients. It is therefore important to develop a rational basis for individualized treatment of NSCLC. Recently, oxaliplatin, a third generation anti-neoplastic platinum-based agent has been explored as a component of therapy for NSCLC. Tumor-specificity and anti-tumor efficacy of oxaliplatin were correlated with active drug influx and OCT-1 expression in colon cancer, but expression of OCT-1 has not been evaluated in NSCLC until now. In this study, we hypothesize that OCT-1 is a major determinant of response to oxaliplatin in lung cancer expressing high levels of the gene and could serve as a predictive biomarker for oxilaplatin-based therapy. We investigated the expression of OCT-1 in lung adenocarcinoma and squamous cell carcinoma specimens using immunohistochemistry (IHC)-based methods. IHC staining was performed on 172 paired specimens on tissue microarrays (TMA) developed in our Thoracic Oncology Program. An anti-human OCT-1 monoclonal antibody and biotinylated anti-rabbit secondary antibody were used for hybridization. The IHC slides were then developed using ABC-DAB procedures and images were acquired using the Aperio ScanScope CS and XT digital slide acquisition systems.

We observed 65 of 91 (71%) adenocarcinomas and 41 of 71 (57%) squamous cell carcinomas to be positive for OCT-1 expression. We identified low levels or absence of OCT-1 expression in the majority of paired normal lung specimens (74%). The OCT-1 levels were significantly higher in both adenocarcinomas and squamous cell carcinomas than in matched normal lung (p<0.001). In addition, the percentage of specimens expressing OCT-1 was significantly higher in adenocarcinomas than in squamous cell carcinomas (p>0.05), suggesting more potential efficacy of oxilaplatin in patients with adenocarcinomas.

In conclusion, OCT-1 was more abundantly expressed in both lung adenocarcinomas and squamous cell carcinomas than in matched normal specimens. The expression status of OCT-1 could serve as a novel predictive biomarker for the selection of patients with NSCLC for oxaliplatin-based chemotherapy. (Supported by NIH grant CA 093708-01A3 and TRDRP grant 17FT-0126)

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 1195.
Abstract 1198: Adaptation to ER stress as a driver of increased expression of Mcl-1 with melanoma progression
Authors: Jiang Chen Chen , Zhuang Li Qing , Dong Li , Throne Rick , Lavis Christopher , Hersey Peter , Zhang Xu Dong
Published: February 2011

Past studies have shown that Mcl-1 expression increases with melanoma progression, which is an important mechanism of resistance of melanoma cells to apoptosis. We show here that up-regulation of Mcl-1 in melanoma is a consequence of adaptation of melanoma cells to endoplasmic reticulum (ER) stress. Exposure of cultured melanoma cells and fresh melanoma isolates to pharmacological ER stress inducers results in up-regulation of Bcl-2 and Mcl-1. Inhibition of Mcl-1 renders melanoma cells more sensitive to ER stress-induced apoptosis even when Bcl-2 is over-expressed, but inhibition of Bcl-2 does not increase the sensitivity to ER stress-induced apoptosis in melanoma cells over-expressing Mcl-1. Up-regulation of Mcl-1 by ER stress involves multiple mechanisms. The transcriptional increase is mediated by the transcription factor ETS-1 downstream of the XBP1 axis of the unfolded protein response, whereas down-regulation of GSK3α is responsible for reduced proteasomal degradation of Mcl-1 in melanoma cells under ER stress. Studies on a large cohort of melanocytic tumor tissue sections revealed that expression of GRP78, a classic marker of ER stress, and expression of LDH5, an indicator of hypoglycemia that is one of the causes of ER stress, correlate with Mcl-1 expression, indicating that the increase in Mcl-1 with melanoma progression in vivo is associated with ER stress. In addition, we demonstrate in this report that induction of apoptosis by ER stress in melanoma cells deficient in Mcl-1 is mediated by the BH3-only proteins PUMA and Noxa, whereas another BH3-only protein Bim does not appear to play a part in melanoma cells as it does in other cell types. In summary, our results indicate that adaptation to ER stress is a driver of up-regulation of Mcl-1 during progression of melanoma, and suggest that strategies aimed at inhibiting the activity of Mcl-1 may be important in the treatment of melanoma.

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 1198.
Abstract 12: Metastatic neuroblastoma cancer stem cells display a mixed phenotype of tumor and niche origin required for survival
Authors: Hansford Loen M. , Morozova Olena , Lipman Tatiana , Blakely Kim , Ohira Miki , Marrano Paula , Angelini Paola , Moffat Jason , Thiele Carol J. , Thorner Paul , Dick John , Nakagawara Akira , Irwin Meredith , Marra Marco , Kaplan David R.
Published: February 2011

Neuroblastoma (NB) is a pediatric tumor of neural crest origin, and is the most common cancer of infancy. 50% of patients have metastases at diagnosis, of which 85% will die after multiple relapses from metastatic disease. We identified tumor-initiating cells (TICs) from bone marrow (BM) metastases of high-risk patients that are propagated as spheres and that have many properties of cancer stem cells, and form NB in mice with as few as 1 cell. To understand patient relapse and disease progression, we compared NB-TICs from BM with those from tumor and brain metastases and SKPs, a normal pediatric stem cell counterpart, by cDNA microarray, flow cytometry, and whole genome shotgun sequencing transcriptome analysis. BM-derived NB-TICs expressed primitive neural crest and neuronal markers as well as hematopoietic markers from primitive, myeloid, and B-cell lineages and contained VDJ gene rearrangements, which are normally associated with B-cell leukemias. Hematopoietic genes were not expressed or expressed at very low levels in tumor-derived sphere lines and a line from an NB brain metastasis, however brain metastasis-derived TICs expressed CD133, while BM-derived NB-TICs did not. Furthermore, we found that shRNA to CD74, which is upregulated in B-cell lymphoma and multiple myeloma and is a therapeutic target for those cancers, induced the rapid death of NB-TICs but not normal SKPs. Interestingly cells co-staining for the hematopoietic markers CD45 or CD74 and the neural neural progenitor marker nestin were found in BM smears of patients with relapsed NB in the bone marrow.

We suggest that metastatic TICs from some tumors adopt resident niche-specific gene expression signatures, which may aid diagnosis and the development of novel treatments. We hypothesize that drugs used for leukemia may be efficacious therapeutics for metastatic NB, and are currently testing this hypothesis in mouse models.

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 12.
Abstract 1212: PIN1 enhances the anti-apoptotic function of survivin in cancer cells
Authors: Cheng Chi Wai , Pang Roberta , Fok Elaine , Kwong Yok Lam , Tse Eric
Published: February 2011

Introduction: The peptidyl prolyl cis/trans isomerase 1 (PIN1) binds the specific motif comprising a phosphorylated serine or threonine residue preceding a proline (pSer/Thr-Pro) in proteins. PIN1 catalyses isomerisation of the prolyl peptide bond, and subsequently induces conformational changes in the substrates and modulates their functions. Through this mechanism, PIN1 is involved in many cellular events, including cell cycle progression, cell proliferation and cell transformation. Our previous work has revealed that PIN1 is over-expressed in hepatocellular carcinomas (HCC) and enhances hepatocarinogenesis. In this study, we investigated the role of the interaction between PIN1 and Survivin, an inhibitor of apoptosis protein (IAP), in tumorigenesis.

Methods and Results: By co-immunoprecipitation experiments, we found that PIN1 interacted with Survivin via the phosphorylated Thr34-pro motif in Survivin. We then further investigated the effects of PIN1 on the anti-apoptotic function of Survivin using flow cytometry by staining the cells with Annexin V and 7-AAD. Both caspase-9 and caspase-3 activities were also detected by flow cytometry. Through these experiments, over-expression of PIN1 in cervical carcinoma HeLa and human nontumorigenic liver MIHA cells were found to suppress the apoptotic response induced by staurosporine through inactivation of caspase-9 and caspase-3. However, the expression of the PIN1 mutants that are catalytically inactive or defective for protein-binding activity did not result in an inhibition of apoptosis. Likewise, the targeted inhibition of PIN1 by small interfering RNA in HeLa and human hepatoma PLC/PRF/5 cells enhanced the apoptotic response induced by staurosporine through caspase-9 and caspase-3 activation. In addition, down-regulation of Survivin by small interfering RNA in PIN1 over-expressing cells abolished the anti-apoptotic effect induced by PIN1, suggesting that the inhibition of apoptosis is mediated through the PIN1-Survivin interaction.

Conclusion: Since phosphorylation of Thr34 in Survivin suppresses pro-caspase-9 activation by increasing the binding between Survivin and pro-caspase-9, our results suggest that PIN1 may further regulate the anti-apoptotic role of Survivin through modulation of its binding to pro-caspase-9.

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 1212.
Abstract 1220: Role of human sLZIP in PMA-induced apoptosis in LNCaP prostate cancer cells
Authors: Kim Yoonseo , Ko Jesang
Published: February 2011

Androgen and the androgen receptor (AR) play important roles in prostate cancer development, and androgen ablation has been the main therapeutic option for the treatment of locally advanced or metastatic prostate cancer. It has been reported that androgen-dependent LNCaP prostate cancer cells undergo apoptosis upon phorbol ester 12-myristate 13-acetate (PMA) treatment. In this study, we investigated whether transcription factor sLZIP is able to protect LNCaP cells from PMA-induced apoptosis. Results from cell morphology, cell cycle and apoptosis assay showed that PMA-induced apoptosis of LNCaP cells was inhibited in cells transfected with sLZIP. We also examined the effect of sLZIP on PARP cleavage and caspase 3 activation. PMA induced PARP cleavage and capase 3 activation, however, cells transfected with sLZIP exhibited reduced cleavage of PARP and caspase 3 in a dose dependent manner. sLZIP induced phosphorylation of ERK and increased cyclin D3expression. OverexpressionofsLZIPattenuatedPMA-mediatedTNF-α induction in LNCaP cells. These results indicate that human sLZIP negatively regulates PMA-induced apoptosis of prostate cancer cells.

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 1220.
Abstract 1222: Down-regulation of MEIS2 leads to apoptosis in human colorectal cancer cells
Authors: Hong Chang Soo , Zhao Xue-Feng , Moon Sung Min , Lee Hye Won , Lee Snag Rok , Park Yong Kyu
Published: February 2011

Three amino-acid loop extension (TALE) homeobox proteins MEIS2 is a transcriptional factor that regulates the expression of large sets of downstream target genes by direct binding to their promoter DNA. MEIS2 controls cell growth and differentiation during embryogenesis and carcinogenesis and is overexpressed in ovarian and pancreas cancer. However, the underlying cellular mechanisms remain largely unsolved. In this study, we report that MEIS2 is aberrantly expressed in colorectal adenocarcinoma cells and is profound proliferative and anti-apoptotic effects against colon cancer cells. In immunohistochemical staining, MEIS2 is positively stained in colorectal adenocarcinoma cells and has strong intensity in its nucleus, compared to normal colonic crypt cells. We found that down-regulation of MEIS2 by specific shRNA inhibited the cell growth (50% decrease as compared with control cultures, P<0.001) of HCT-116 colon cancer cell, due to an increase apoptosis (from 3% in control cultures to 65% after 72 hour from transient transfection of MEIS2 shRNA, P<0.001) as determined by MTT and FACScan apoptosis assay. Knockdown of MEIS2 by specific shRNA transfection reduced the expression of Akt, and decreased the phosphorylation of its downstream target proteins such as forkhead transcription factors (FoxO) and p27, and increased the nuclear localization of nonphosphorylated FoxO and p27, which result in activation of apoptosis. In summary, our data indicate that MEIS2 promotes cell growth and plays critical role as novel anti-apoptotic factor through regulation of Akt expression within Akt/PKB signaling pathway in colorectal 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 1222.
Abstract 1236: Interferon consensus sequence binding protein (ICSBP)-induced cell proliferation mediated by TGF-β signaling pathway and MAPKs activation in HL-60 cells
Authors: Park Byung-Kiu , Kim Hee Jung , Sung Jee Young , Kim Yong Nyun
Published: February 2011

Interferon consensus sequence binding protein (ICSBP), also known as IRF-8, is a member of the interferon (IFN)- regulatory factors (IRFs) induced by IFN-γ. ICSBP is mostly associated with differentiation of hematopoietic cells and known as a tumor suppressor. ICSBP contains the conserved transactivation domain which is shared by Smad4, suggesting a possible connection of IRF and TGF-β signaling. To test this connection, ICSBP was stably expressed in acute promyelocytic leukemia cell line, HL-60, that expresses little ICSBP. Although TGF-β receptor expression was undetectable in HL-60 cells, the expression of ICSBP in HL-60 (ICSBP-HL-60) resulted in an increased TGF-β receptor expression. Interestingly, even without TGF-β stimulation, both Smad and non-Smad pathways were activated in ICSBP-HL-60 cells compared with Mock-HL-60 cells, indicating ligand-independent activation of TGF-β receptor signaling. In addition, ICSBP expression augmented cell growth and suppression of ICSBP using siRNA specific for ICSBP abolished TGF-β receptor expression and attenuated cell growth. Consistently, reduction of TGF-βRI using siRNA or TGF-β receptor inhibitor, SB431542, also attenuated growth of ICSBP-HL-60 cells compared with Mock cells. Furthermore, MAPK pathways appeared to be involved in ICSBP-mediated cell growth because MAPKs are activated in ICSBP-HL-60 cells and specific inhibitor of this pathway was able to decrease the growth of ICSBP-HL-60 cells more sensitively than Mock-HL-60 cells. Suppression of ICSBP by siRNA effectively inhibited phosphorylation of MAPKs. All these data suggest that ICSBP up-regulates TGF-β receptor, activates ligand-independent TGF-β mediated signaling pathways, such as MAPKs activation, leading to cell proliferation. This study provides a new function of ICSBP in the regulation of cell proliferation

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 1236.
Abstract 1241: Tcf7, a mediator of Wnt/β-catenin signaling, is upregulated in mammary glands of BK5.ATF3 mice, without prior development of mammary tumors
Authors: Yan Leqin , Powell K. Leslie , MacLeod Michael C.
Published: February 2011

We previously showed that in a transgenic model (BK5.ATF3) in which the transcription factor ATF3 is overexpressed in cytokeratin 5 (CK5)-expressing cells, parous female mice develop mammary carcinomas with about 70% incidence between 6 months and one year of age. In these mice, ATF3 is overexpressed in cell nuclei in the myoepithelial cell layer (basal layer) of normal mammary glands, and in the basal cell layers of the tumors, in addition to expression in epidermis and other CK5-expressing epithelia. Non-parous, female transgenic mice exhibited a high incidence of squamous metaplastic lesions in the mammary glands between 14 and 32 weeks of age. These lesions showed dysregulation of cytokeratin expression, but no other molecular defects in non-parous mammary glands have been described. Several genes in the EGFR-MAPK pathway and in the Wnt/β-catenin pathway are misregulated in the mammary tumors that arise in parous transgenic mice. When expression of these genes was compared between normal mammary glands from transgenic and non-transgenic mice, the only genes that showed significant differences were Lef1 and Tcf7, two members of the family of transcription factors that dimerize with β-catenin to mediate Wnt-induced transcription. These genes were overexpressed by 2.5-3-fold in the transgenic mammary glands. A mouse mammary tumor cell line, EMT6, was found to have high levels of expression of both Atf3 and Tcf7, but not Lef1. Expression of Atf3 was knocked down by 97% in EMT6 cells 48 hrs post-treatment with Atf3-specific siRNA. This resulted in about a 70% decrease in expression of Tcf7, and a similar decrease in expression of cJun, a known target of Wnt/β-catenin signaling. This is consistent with the possibility that Tcf7 is a target gene for Atf3, and may be involved in the early stages of tumorigenesis in this model.

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 1241.
Abstract 1247: Transcriptional regulation of VEGF by WT1 and AR in prostate cancer
Authors: Eisermann Kurtis , Reese Jennifer L. , Broderick Carly J. , Fraizer Gail C.
Published: February 2011

Identifying molecular mechanisms that influence prostate cancer progression is central to understanding how this disease develops. Angiogenesis is key for cancer development and growth as tumors require new blood vessels for nutrients and oxygen. Vascular endothelial growth factor (VEGF) is the most important signaling protein involved in angiogenesis and is up-regulated by oncogene expression among other things. Previous studies have shown that the zinc finger transcription factor WT1 (Wilms tumor 1) transcriptionally regulated VEGF in multiple cell lines. Additionally, WT1 activation of VEGF required an intact zinc finger domain and was mediated by DNA binding. Potential WT1 binding sites were identified in the VEGF promoter using in silico approaches and shown to bind WT1 protein using electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assays. Luciferase reporter assays showed increased activation of the VEGF promoter by over-expression of WT1. Additional factors known to induce VEGF expression are growth factors, hypoxia and hormones. How androgen regulates VEGF transcription is unknown, but we have identified two potential androgen receptor (AR) binding sites within 2kb of the transcriptional start site and demonstrated reporter activation by R1881 treatment of transfected LNCaP cells. This study examined the effect of mutating selected WT1 and AR binding sites in the VEGF promoter to determine whether they were responsible for activation of VEGF transcription. Mutating these binding sites by site-directed mutagenesis significantly decreased activation of the VEGF promoter by WT1 and the androgen analog R1881 in LNCaP cells. As predicted, treatment with the anti-androgen casodex similarly eliminated hormone response of the VEGF promoter, suggesting that androgens are functioning via androgen signaling. This was confirmed in vivo by both western blot analysis of VEGF protein and quantitative real-time PCR measurement of VEGF mRNA in cells treated with casodex. This is important because androgen signaling is vital to normal prostate development and malignant prostate growth. Overall these results show that both WT1 and AR transcriptionally regulate VEGF expression in prostate cancer cells and these factors directly bind and activate the promoter in vivo. Understanding how angiogenesis is regulated will lead to improved therapy and prevention of prostate cancer progression.

This work was supported by NIHR15CA11360 (GF), Sigma Xi GIAR220034 (KE), and GSSKSU (KE)

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 1247.
Abstract 125: The DNp73 isoform provides novel insights into the regulation of p53 by BRCA1 to favor cell survival over cell death
Authors: Scata Kimberly A. , El-Deiry Wafik S.
Published: February 2011

BRCA1, the first breast cancer susceptibility gene to be identified, remains an enigma. Many roles have been ascribed to BRCA1 including DNA repair, cell cycle checkpoint activation, transcriptional regulation and chromatin remodeling. We have previously established that BRCA1 overexpression induces the stabilization of p53 without activating pro-apoptotic targets of p53, and that BRCA1 can protect cells from DNA damage-induced p53-dependent cell death. This seems to be specific to pro-apoptotic targets because p53 is still able to activate target genes involved in cell cycle arrest or DNA repair. In our efforts to understand this phenomenon, we have found that BRCA1 induces many of the same post-translational modifications that DNA damage induces including key phosphorylations and the acetylation of lysine 120 of p53, known to be important for apoptosis. Moreover, BRCA1-stabilized p53 localizes to the promoter regions of pro-apoptotic target genes whose expression is not upregulated. We hypothesized that p53 family members p63 and/or p73 may regulate the selectivity of p53 action in response to BRCA1. Unexpectedly, we found that BRCA1 upregulates DNp73 expression, an isoform of the p53 family member p73 lacking the transactivation domain, that has been previously implicated in cell survival. This observation has important implications for understanding the selectivity of p53-dependent transcription as well as the tumor suppressive action of BRCA1 that favors DNA repair over cell death. Our ongoing studies are evaluating the effects of the BRCA1-induced DNp73 isoform on interactions between p53 and p53 family members that regulate the genes involved in cell fate. These findings unravel a novel mechanism by which the selectivity of p53-dependent gene expression is regulated BRCA1 and further unravels the complex nature of these p53 and BRCA1 tumor suppressor pathways.

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 125.
Abstract 1255: Protein kinase C epsilon (PKCε) as a target for lung cancer treatment: Essential role of PKCε in tumor growth and metastatic dissemination of non-small cell lung cancer (NSCLC) cells
Authors: Caino M. Cecilia , Haber Cynthia C. Lopez , Kim Jeewon , Kazanietz Marcelo G.
Published: February 2011

Protein Kinase C epsilon (PKCε), a member of the PKC family of phorbol ester/diacylglycerol receptors, has been recently shown to be highly overexpressed in non-small cell lung carcinomas (NSCLCs) compared to normal lung epithelium. The fact that several of the most common genetic alterations in lung cancer, such as EGFR, K-Ras, or PI3K mutations, could signal through PKCs, led us to hypothesize that PKCε may contribute to lung tumorigenesis. We found that the expression of PKCε was markedly up-regulated in NSCLC cell lines (H358, H441, H322, H1299, and A459) relative to immortalized non-tumorigenic cells (HBEC-3). Stable knockdown of PKCε in NSCLC cells using shRNA lentiviruses impaired cell survival in response to serum starvation, as well as it increased the sensitivity to senescence induction by phorbol esters. Moreover, anchorage-dependent and -independent growth were markedly reduced in PKCε-depleted NSCLC cells. Notably, silencing of PKCε-expression in NSCLC impaired motility in response to PMA, FBS or TGFα. Treatment of NSCLC cells with PMA led to a significant enhancement in cell motility as well as it promoted ruffle formation. Both ruffle formation and migration were mediated by PKCε. Moreover, PMA caused a marked enhancement in Rac1-GTP levels in NSCLC cells, but this effect was lost when PKCε was knocked down using RNAi. To address the relevance of our findings in an in vivo setting, we examined the requirement of PKCε in tumorigenesis and metastasis. Remarkably, PKCε-depleted H358 cells have impaired tumorigenic potential when inoculated into nude mice. At early time points during tumor formation (6-15 days) we observed a significant increase in apoptotic cells in PKCε-depleted H358 xenografts compared to controls, as determined by TUNEL. These in vivo results were validated pharmacologically by means of a specific inhibitor of PKCε translocation/activation. Indeed, delivery of the specific PKCε inhibitor

εV1-2 by osmotic minipumps greatly reduced the rate of tumor growth in nude mice. Finally, we sought to study whether PKCε is implicated in tumor dissemination. Using a tail vein injection assay, we found that stable depletion of PKCε essentially impaired the appearance of lung metastatic foci in nude mice. In summary, our results established PKCε as a key mediator of lung tumorigenesis and provided strong evidence that elevated PKCε levels in NSCLC cells play a key role in metastatic dissemination. We speculate that PKCε is an attractive pharmacological target for lung cancer treatment.

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 1255.
Abstract 1264: Extracellular galectin-3 induces tumor-specific apoptosis through the interaction with highly glycosylated cell surface β1 integrin
Authors: Lee Sok-Hyong , Zerrouqi Abussamad , Devi Saroja , Van Meir Erwin G.
Published: February 2011

Galectins are a family of animal lectins, which bind beta-galactose moieties. Gal3 is the only chimeric family member of Gals and consists of a collagen-like N-terminal region and a C-terminal carbohydrate recognition domain (CRD), which has binding affinity for galactose and N-acetyl-lactosamine. Gal3 is known to be secreted through Golgi apparatus-independent alternative pathway. Previously, we found that stimuli-induced p53 activation induced bystander killing effect against adjacent cells. This p53 bystander effect is mediated through the secretion of Gal3 with the requirement of p53-dependent induction of tumor suppressor activated pathway-6 (TSAP6). We found the apoptotic activity of extracellular Gal3: a variety of tumor cell types (malignant glioma, breast, colon, prostate, lung) are sensitive to Gal3-mediated killing, while normal cells (fibroblast, endothelial) are not affected. We demonstrated that conditional sGal3 induction (tet-on system) in glioma xenografts strongly inhibits tumor growth without overt toxicity in mice. We found that Gal3-mediated tumor cell killing was neutralized with lactose, a Gal3-CRD binding ligand, and Gal3-mediated apoptosis was inhibited by treatment with a caspase 9 inhibitor (LEHD) but not by a caspase 8 inhibitor (IETD). Gal3 interacts with several glycosylated cell surface proteins. We identified that Gal3-induced tumor specific apoptosis is mediated through Gal3-beta1 integrin binding through GST-Gal3 pulldown assay, cell membrane-specific Gal3-beta1 integrin co-immunoprecipitation, and beta1 integrin siRNA neutralization. Further, the treatment of N-glycosylation inhibitor (tunicamycin) interfered with Gal3-beta1 integrin interaction whereas, O-glycosylation inhibitor (benzyl-O-N-acetyl-D-galactosamine) increased N-glycosylation of beta1 integrin as well as consequent Gal3-beta1 integrin interaction and Gal3-mediated apoptosis. In summary, our data demonstrate that extracellular Gal3 interacts with tumor cells through highly-glycosylated cell surface beta1 integrin through its CRD binding and consequently induces apoptosis. These findings are important for our understanding of tumor-specific killing of Gal3 and suggest that Gal3 can be exploited as a therapeutic agent for cancer.

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 1264.
Abstract 1281: Endothelial cells promote the cancer stem cell phenotype of human colorectal cancer cells
Authors: Lu Jia , Bose Debashish , Zhou Yunfei , Gaur Puja , Samuel Shaija , Fan Fan , Xia Ling , Ye Xiang-Cang , Sceusi Eric , Tozzi Federico , Mani Sendurai A. , Ellis Lee M.
Published: February 2011

Background: Cancer stem cells (CSC), which possess the properties of self-renewal, multi-potential differentiation and tumor initiation, can arise either from normal stem cells or due to the influence of the tumor micro-environment. This study was done to determine the potential role of endothelial cell (EC) derived paracrine factors on promoting the CSC phenotype in human colorectal cancer (CRC) cells.

Methods: RFP-labeled human CRC cells HCT116 were co-cultured with RF24 ECs (immortalized HUVECs) under low serum conditions. After FAC-sorting for the CRC cells, the CSC population was analyzed by 1) the Aldefluor assay, 2) sphere forming ability, and 3) Western blot analysis for the CSC markers CD133 and CD44. In addition, low serum condition media obtained from either ECs or parental CRC cells (control), was added to CRC cells to determine its effect on the CSC phenotype. In order to characterize the apoptotic effect of EC derived factors on CRC cells, we performed Annexin V staining by FACS, PARP cleavage, Caspase 3 cleavage, and Bcl2 levels by Western blotting. Using MTT assay, we determined the effect of EC derived factors on chemo-sensitivity of CRC cells exposed to 5-FU.

Results: Co-culturing of CRC cells with ECs markedly increased the ALDH-positive population from 5% to 18%, and the sphere forming ability by more than 4-fold in CRC cells. In addition, CRC cells also displayed increased CD133 (6-fold) and CD44 (4-fold) protein levels. Furthermore, this effect could be mimicked simply by co-culturing CRC cells with conditioned media obtained from ECs. Similarly, treatment of CRC cells with conditioned medium from ECs significantly increased the ALDH-positive population from 4% to 8%, sphere forming ability >3-fold, and the expression of CD133 (7-fold) and CD44 (6-fold). Conditioned medium from ECs, concomitantly decreased spontaneous apoptosis in CRC cells as demonstrated by a 3-fold decrease in Annexin V-positive population (21% to 7%), down-regulation of cleaved PARP and Caspase 3, and up-regulation of Bcl2. CRC cells exposed to EC conditioned medium also displayed decreased sensitivity to 5-FU [>10-fold increase of the IC50 from 2.0 μg/ml to 22.3 μg/ml (p<0.05)].

Conclusions: Exposure of CRCs to ECs increased CSC properties, and decreased spontaneous apoptosis. Since, this effect can also be mimicked by the conditioned media, additional experiments to identify and validate the soluble factors that mediate this effect are in progress.

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 1281.
Abstract 1307: Downregulation of vascular endothelial cell growth inhibitor (VEGI; TNFSF15) expression in endothelial cells by interferon-γ
Authors: Lu Yi , Chen Li , Li Lu-Yuan
Published: February 2011

Vascular endothelial growth inhibitor (VEGI) is a member of the tumor necrosis factor superfamily (TNFSF15) produced largely by endothelial cells. It functions as an endogenous inhibitor of neovascularization. We showed previously that VEGI inhibits angiogenesis by enforcing a growth arrest on quiescent endothelial cells while inducing apoptosis in proliferating endothelial cells. We also found that VEGI gene expression is down-regulated in growing endothelial cells in vitro as well as in tumor vasculatures. Here we show that, among a variety of inflammation-modulating cytokines we evaluated, interferon-γ (IFNγ) can down-regulate VEGI expression in human umbilical vein endothelial cells in a dose- and time-dependent manner. Neutralizing antibodies of IFNγ receptor 1 restores VEGI expression. IFNγ modulation of VEGI gene expression is mediated by unphosphorylated STAT1 rather than phosphorylated STAT1. These findings are consistent with the view that down-regulation of VEGI gene expression in endothelial cells by IFNγ is a prerequisite for the initiation and continuation of angiogenesis during inflammation.

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 1307.