bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2904: Docking-based virtual screening of Chk1 inhibitors
Authors: Li Yan , Kim Dong Joon , Bode Ann M. , Dong Zigang
Published: June 2012

Checkpoint 1 (Chk1), which is required for checkpoint arrest, is activated in response to DNA damage through the ATM/ATR pathway, and plays an essential role in the regulation of the G2/M checkpoint. Inhibition of Chk1 should abrogate this checkpoint in a manner relevant to the treatment of cancer, and may also induce apoptosis. Selective Chk1 inhibitors should also prove useful to study the regulation of the G2/M checkpoint. Several Chk1 inhibitors are already known, including natural products. UCN-01, a Chk1 inhibitor and staurosporine derivative, is undergoing clinical trials as an anticancer agent. A limited number of additional Chk1 inhibitors have been reported; however, a need to identify more drug-like Chk1 inhibitors is clear. This should be facilitated by the structural information available for the Chk1 kinase domain. The first crystal structure of the kinase domain of human Chk1 has been reported at 1.7Å resolution in 2000. After that, 46 co-crystal structures of Chk1 and inhibitors have also been solved. This puts docking-based virtual screening of Chk1 inhibitors on firm footing. Molecular docking is a very popular method employed to investigate molecular associations and is particularly useful in the drug discovery arena to study the binding of small molecules (ligands) to macromolecules (receptors). To deal with the binding event in a realistic manner, we took into account the conformational flexibility of the ligand and the flexibility of the receptor. In this work, a large collection of crystallographic structures of Chk1 was employed to determine if multiple-structure docking performs better than single-structure docking. Known ligands were used to test the success of docking in binding mode prediction and virtual screening enrichment factors. A chemical library with more than 60,000 compounds was docked into the selected Chk1 structures, and the top-ranked compounds were then tested in experiments.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2904. doi:10.1158/1538-7445.AM2011-2904
Abstract 2905: The HMGA1-MMP-2 pathway in poorly differentiated uterine tumors
Authors: Hillion Joelle , Cello Francescopaolo Di , Huso David , Resar Linda
Published: June 2012

Uterine cancer is the most common cancer of the female genital tract and the fourth most frequent cause for cancer death in women in the United States. Thus, further study has the potential to significantly benefit women's health worldwide. Despite the high prevalence of this disease, the molecular events that lead to uterine cancers are poorly understood. We are studying the role of the high mobility group A1 (HMGA1) gene in the pathogenesis of uterine cancer. The HMGA1 gene encodes the HMGA1a and HMGA1b chromatin remodeling proteins, which function in modulating gene expression. In a previously published pilot study, we showed that HMGA1 is overexpressed in poorly differentiated uterine cancers, but not in normal uterine tissue, benign tumors, or most low-grade neoplasms of the uterus. We also engineered HMGA1 transgenic mice and found that all females develop uterine sarcomas by 9 months of age. In lung cancer, we reported that HMGA1 induces expression of the matrix metalloproteinase-2 (MMP-2) gene, but only in poorly differentiated tumors, suggesting that this pathway could drive tumor progression and a poorly differentiated state. Here, we investigated the role of the HMGA1-MMP-2 pathway in the pathogenesis of uterine tumorigenesis. Using a genetic approach, we crossed our HMGA1 transgenic mice with mice that are null for the MMP-2 gene. We found that the uterine tumors were significantly smaller in the HMGA1 transgenic mice that are also null for MMP-2. To determine if this pathway is involved in human uterine tumorigenesis, we assessed expression of the HMGA1 and MMP-2 genes in primary, human uterine tumors. We studied: 1.) endometrioid tumors, which are derived from the endometrial epithelium and tend to be low-grade (well-differentiated), 2.) serous tumors, which are also derived from the endometrial epithelium, but tend to be high-grade (poorly differentiated), and, 3.) carcinosarcomas (or mixed malignant, mullerian tumors), which are derived from the endometrial epithelium and stroma and tend to by high-grade. We found that HMGA1 expression was highest in the high-grade serous tumors and carcinosarcomas, with lower levels in the endometrioid tumors. Moreover, we found that there was a significant correlation between HMGA1 and MMP-2, but only in the high-grade carcinosarcomas and serous tumors. Taken together, our studies demonstrate that HMGA1 induces expression of MMP-2 during uterine tumorigenesis and suggest that this pathway is important in aggressive, poorly differentiated tumors. Although further studies are needed, our findings also suggest that the HMGA1-MMP-2 pathway could serve as a rational therapeutic target in poorly differentiated uterine cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2905. doi:10.1158/1538-7445.AM2011-2905
Abstract 2910: Oncogenic role of MUC1 in context of TGF-β production and signaling
Authors: Nath Sritama , Rao Shanti , Tinder Teresa L. , Gendler Sandra , Mukherjee Pinku
Published: June 2012

Introduction: Pancreatic cancer is the fourth leading cause of cancer-related death in United States. Most human pancreatic cancers overexpress MUC1, which is associated with high metastasis and poor prognosis of the disease. We have recently shown that MUC1 initiates the process of epithelial to mesenchymal transition (EMT) and promotes metastasis in pancreatic cancer cells via signaling through the seven tyrosines present in its cytoplasmic tail (CT) motif. When the 7 tyrosines were mutated to phenylalanine, EMT was completely abrogated. Since TGF-β has been extensively studied as a mediator of EMT and invasion in pancreatic cancer, we hypothesized that MUC1 may be inducing its effect through regulating TGF-β production and signaling. We also aimed at identifying the specific tyrosine residue(s) responsible for MUC1 induced EMT. Method: We made the following plasmids containing a) full length MUC1 (MUC1), b) all 7 tyrosine residues of MUC1 CT were mutated to Phenylalanine (Y0), c) Y6 (Tyrosine 6 present, the rest replaced by Phenylalanine), d) Y7 (Tyrosine 7 present, the rest replaced by Phenylalanine), e) Y2,5 (Tyrosine 2 and 5 present, the rest replaced by Phenylalanine), f) Y3,6,7 (Tyrosine3, 6 and 7 present, the rest replaced by Phenylalanine) and g) empty vector (Neo). These plasmids were stably expressed in human pancreatic cancer cell lines, BxPC3 and Su86.86 that express low levels of endogenous MUC1. We first assessed the levels of functional TGF-β secreted by BxPC3 Neo, MUC1 and Y0 cells as well as the levels of TGF-βI and II receptors. We then treated these cells with exogenous human recombinant TGF-β and assessed both, the process of EMT and the capacity to invade through growth-reduced matrigel. Results: We found that BxPC3.MUC1 cells produced strikingly larger amounts of TGF-β compared to BxPC3 Neo and Y0 cells. We also found that MUC1-expressing cells became more invasive in response to exogenous TGF-β while the Y0 cells did not respond to exogenous TGF-β although the receptor levels were similar in all three cell lines. However, none of the mutants of MUC1 CT could rescue the function of full length MUC1, in the context of induction of EMT. Conclusion: 1) We believe that TGF-β mediated EMT and TGF-β signal propagation requires the tyrosine residue of MUC1, which is evident from the data that mutating the tyrosine residues in MUC1CT abrogates TGF-β mediated EMT in these cells. 2) Induction of EMT by MUC1 is a complex process, which involves a particular combination of tyrosine residues of MUC1.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2910. doi:10.1158/1538-7445.AM2011-2910
Abstract 2912: Protein pathway activation mapping of leukemia-associated JAK1 mutants
Authors: Fodale Valentina , Flex Elisabetta , Stellacci Emilia , Policicchio Eleonora , Deng Jianghong , Calvert Valerie S. , Liotta Lance A. , Petricoin Emanuel F. , Tartaglia Marco
Published: June 2012

The Janus kinase 1 (JAK1) is a cytoplasmic tyrosine kinase that noncovalently associates with a variety of cytokine receptors and plays a nonredundant role in lymphoid cell precursor proliferation, survival and differentiation. We documented that somatic mutations in JAK1 occur in individuals with acute lymphoblastic leukemia (ALL) (Flex et al. 2008, J Exp Med 205:751-8). JAK1 mutations were more prevalent among adult subjects with T-cell precursor ALL, where they accounted for approximately 20% of cases, and were associated with poor response to therapy and overall prognosis. In order to understand the molecular and cellular mechanisms by which aberrant JAK1 function contribute to leukemogenesis we analysed the signaling architecture of BaF3 cell lines stably expressing a series of leukemia-associated JAK1 gene mutations by Reverse Phase Protein Microarray (RPMA). We utilized BaF3 cell lines, stably expressing human wild type JAK1 and five ALL-associated mutants bearing missense mutations in different domains of the kinase. Cells were serum starved for 16 hours and lysed before and after induction with IL-3 for 20 minutes. RPMA analysis was performed to determine the phosphorylation/activation state of 102 different key signalling proteins involved in the major intracellular signal transduction pathways. Results showed that intacellular signaling downstream JAK/STAT is differentially modulated between cell lines expressing the different ALL-associated JAK1 mutants. Statistical analysis revealed that mutations affecting the pseudo-kinase and in the kinase domains resulted in a different phosphorylation pathway network compared to mutations involving the FERM and SH2 domains.

Our results support a model in which mutations affecting distinct domains of JAK1 have a differential perturbing effect on JAK1 function and signalling. Characterization of the consequences of leukemia-associated JAK1 mutants on the protein signalling network may help to identify new therapeutic targets for ALL. The present findings further document that functional protein pathway activation mapping using RPMA represents a powerful approach to uncovering mutation-driven events, and elucidating the specific events associated with different mutations within a single protein.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2912. doi:10.1158/1538-7445.AM2011-2912
Abstract 2919: Unexpected role of CRAF in tumor cell mitosis revealed by an allosteric inhibitor
Authors: Mielgo Ainhoa , Murphy Eric , Huang Miller , Seguin Laetitia , Weis Sara , Cheresh David
Published: June 2012

RAF kinases regulate fundamental cellular processes such as proliferation and survival and can be mutated or dysregulated in tumors. Although, RAF inhibitors show clinical activity in some cancer patients, recent reports have shown that ATP-competitive RAF inhibitors can enhance the proliferation of RAS driven tumors by promoting B- and CRAF dimerization and phosphorylation of CRAF on serine 338. Recently, we characterized an allosteric inhibitor of RAF that disrupts RAF dimerization, CRAF serine 338 phosphorylation and tumor growth in mice. Here, we report the surprising finding that phospho-S338 CRAF localizes to the mitotic spindle of proliferating tumor cells in vitro and in biopsies from cancer patients. Importantly, treatment of these cells with an allosteric, but not an ATP-competitive inhibitor of RAF, prevents CRAF localization to the mitotic spindle and causes cell cycle arrest at pro-metaphase in a MAPK-independent manner. Mechanistically, phospho-S338 CRAF interacts and co-localizes with active Polo-like kinase 1 (Plk1) at the mitotic spindle of proliferating cells. In fact, allosteric or genetic inhibition of CRAF inhibits Plk1 activity and its accumulation at the kinetochores causing pro-metaphase arrest. These findings define an unexpected role for RAF in mitosis, revealing a novel mechanism for RAF as a therapeutic target in oncology.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2919. doi:10.1158/1538-7445.AM2011-2919
Abstract 2925: Protein pathway activation mapping of the NCI60 cell line series: Discovery of lineage-independent protein biomarkers for drug sensitivity/resistance prediction
Authors: Federici Giulia , Fodale Valentina , Deng Jianghong , De Maria Ruggero , Liotta Lance A. , Petricoin Emanuel F. , Wulfkuhle Julia D.
Published: June 2012

The NCI-60 cell lines are a set of human tumor cell lines derived from different organ sites including brain, blood and bone marrow, breast, colon, kidney, lung, ovary, prostate and skin. These cell lines have been characterized through genetic screening, such as chromosome karyotyping, gene expression analysis, protein expression and sequencing and are likely the most well-characterized cell lines in the world. Through the use of Reverse-Phase Protein Microarrays (RPMA) we performed an in-depth analysis of signal pathway activation, measuring the activation state of nearly 200 key signaling proteins representative of pathways involved in proliferation, survival, inflammation, adhesion, and migration. This broad-based screening allowed the characterization of activated intracellular signaling pathways and identified 11 clear subclasses based on molecular architectures which transcended cell line lineage. Statistical analysis of signaling architecture of the most drug-resistant and sensitive lines, defined by publicly available GI50 data was performed for a number of molecularly targeted-therapeutics. We identified novel and previously known protein biomarkers that correlated with resistance and sensitivity to these inhibitors, which indicated the potential for functional signaling analysis of the cell lines to produce biomarkers that could be useful in vivo if extensively validated. Our goal is to be able to assign a specific protein signaling architecture for each specific drug sensitivity profile such that predictive markers can emerge in a biochemically-driven fashion.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2925. doi:10.1158/1538-7445.AM2011-2925
Abstract 2931: Zerumbone, a phytochemical from asian ginger inhibits JAK/STAT pathway, growth, apoptosis and increase taxol sensitivity of hormone refractory prostate cancer cells
Authors: Chakraborty Arup , Jorvig Jessica
Published: June 2012

Objectives: Prostate cancer (PCa) is the most common cancer incidence and the second cancer death in U.S. men. In 2010, an estimated 217,730 new cases of PCa will be diagnosed, and 32,050 people will die from the disease in USA alone. IL-6 is strongly associated with malignant phenotype of PCa. IL-6 potently activates signal transducers and activators of transcription 3 (STAT3), a member of the family of latent cytoplasmic transcription factors that transmit signals from the cell membrane to the nucleus. STAT3 activation is known to promote tumor cell proliferation, survival, angiogenesis, and metastasis in vivo. In PCa, IL-6 strongly promotes mitogenic and angiogenic signaling through phosphorylation of Jak2 and then STAT3. Zerumbone, a relatively new anticancer component isolated from Zingiber zerumbet Smith has not been previously evaluated for prostate cancer.

AIM: 1. Determine the activity and mechanism of zerumbone on hormone refractory prostate cancer cells. 2. Evaluate the combined effect of taxol (paclitaxel) with zerumbone in killing hormone-refractory prostate cancer cells.

Methods and Results: The apoptosis and cytotoxic effect of zerumbone were determined by PARP cleavage (western blot) and MTS assay in DU145 cells. Zerumbone induced cytotoxicity with IC50 = 24 µM and significant PARP cleavage. Zerumbone (25 μM) potently inhibited constitutively activated Jak2/STAT3 phosphorylation and IL-6-stimulated STAT3 phosphorylation in DU145 cells as determined by western blots. Zerumbone (25 μM) also blocked expression of STAT3 dependent antiapoptotic proteins Bcl-xL. Interestingly, zerumbone even at a higher dose like 50uM has no effect on p65 subunit phosphorylation of NFkB and MAP kinase phosphorylation indicating a relative specificity. Next, we determined the combined effect of zerumbone and taxol in DU145 cells. Zerumbone as low as 10µM (4% cytotoxicity) synergistically increased cytotoxicity of taxol (5 nM, 7% cytotoxicity) to 25% cytotoxicity when combined. Zerumbone (10µM) also decreased IC50 of taxol from 12 nM to 7 nM.

Conclusion: We showed that zerumbone is a potential therapeutic agent in prostate cancer by effectively blocking Jak2/STAT3-mediated signaling pathways and in addition, zerumbone combining with taxol based cytotoxic therapies may offer encouraging strategies for combating hormone-refractory prostate cancers.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2931. doi:10.1158/1538-7445.AM2011-2931
Abstract 2940: A role for PAX8 in promoting tumor cell type-specific proliferation through positively regulating E2F1 transcription and stabilizing RB protein
Authors: Eccles Mike R. , Li Caiyun G. , Nyman Jennifer E. , Braithwaite Antony W.
Published: June 2012

The PAX proteins are tissue-specific developmental regulators required for fetal development. In most cases, PAX expression is silenced post-natally, although it is maintained in some adult tissues. Several PAX proteins interact with RB, a key protein involved in cell cycle regulation. The phosphorylation of RB leads to the mobilization of the master regulator of cell cycle progression, E2F1. It is presently unknown how the growth and differentiation of a tissue is coordinated in a tissue-specific manner with the regulation of the cell cycle in order to facilitate terminal differentiation, or how this process might be disrupted in cancer. PAX gene dysregulation is observed in many cancer types with PAX8 being frequently expressed in renal cell carcinoma (RCC), bladder, ovarian and thyroid cancer cell lines. We investigated the effect on the RB-E2F1 pathway of knocking-down PAX8 using siRNAs in cell lines corresponding to these four cancer types. Knockdown of PAX8 led to a striking reduction in E2F1 mRNA and protein in cancer cell lines, as well as a reduction in levels of E2F1 target genes, including cyclin A2 (CCNA2) and CDC6, induction of a G1/S cell cycle arrest, the onset of cellular senescence, and a proteosome-dependent destabilization of RB protein, with RB1 mRNA levels remaining unaffected. Using ChIP and luciferase reporter assays we showed that PAX8 binds to and transcriptionally regulates the E2F1 promoter. These data suggest that the expression of PAX8 is associated with transcriptional activation of E2F1 and stabilization of RB protein in cancer cell lines. During development in normal tissues we presume that expression of PAX8 coordinates cell proliferation with tissue-specific growth. In cancer, however, the expression of PAX8 continues unabated, and PAX8 silencing leads cancer cells to undergo cell cycle arrest, and subsequently to become senescent.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2940. doi:10.1158/1538-7445.AM2011-2940
Abstract 2954: The transient receptor potential V6 (TRPV6) calcium channel in breast cancer cell lines and clinical breast cancer tissues
Authors: Peters Amelia , Simpson Peter , Bassett Jonathon , Lakhani Sunil , Roberts-Thomson Sarah , Monteith Gregory
Published: June 2012

TRPV6 is an important calcium channel, which is highly selective for Ca2+ and has high basal activity. In prostate tumors TRPV6 over-expression is linked to enhanced invasiveness and proliferation. While some studies have demonstrated that TRPV6 is over-expressed in clinical breast cancer samples and studies from our laboratory have shown that TRPV6 mRNA is up-regulated in a sub-set of breast cancer cell lines, a link between TRPV6 expression and breast cancer aggressiveness has not been demonstrated. Furthermore, the mechanism for the over-expression of TRPV6 in breast cancer is unknown. To explore TRPV6 gene amplification as a possible mechanism for over-expression, we used a real-time PCR copy number assay and identified a sub-set of breast cancer cells lines that exhibited increased TRPV6 gene copy number. To assess the clinical relevance of this finding, we initially used a high throughout, clinically driven approach to examine TRPV6 gene amplification in breast tumors. Our findings showed that the mechanism of TRPV6 over-expression is due to, at least in part, gene amplification and that gene amplification may be pronounced in breast cancer subtypes with a poor prognosis. In clinical breast tumor samples, microarray based comparative genomic hybridization (aCGH) showed that the TRPV6 gene region (7q34) is gained in 0/16 invasive lobular carcinomas and in 11/35 high grade invasive ductal carcinomas. When stratified by estrogen receptor status, 7/13 estrogen receptor negative, high grade invasive ductal carcinomas harbored a gain in this region. To determine whether the copy number increase observed at the gene region 7q34 specifically corresponded with amplification of the TRPV6 gene, we performed chromogenic in situ hybridization (CISH) using a probe specific for the TRPV6 gene and a breast cancer tissue microarray. Our findings suggest that a sub-set of breast cancers may be characterized by TRPV6 gene amplification.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2954. doi:10.1158/1538-7445.AM2011-2954
Abstract 2957: Mutations of Cyclin D1 / beta-catenin are coexistent with mutations in Ras-PI3K pathway genes in endometrial cancer
Authors: Ikeda Yuji , Oda Katsutoshi , Nakagawa Shunsuke , Shoji Keiko , Kashiyama Tomoko , Ishikawa Shumpei , Yamamoto Shogo , Tanikawa Michihiro , Hiraike Osamu , Aburatani Hiroyuki , Yano Tetsu , Taketani Yuji
Published: June 2012

Introduction: Mutant beta-catenin is translocated to nucleus, and induces Cyclin D1 expression. Cyclin D1 cooperates with CDK4/6 to accelerate cell cycle from G1 to S phase. In addition to canonical Wnt signaling pathway, beta-catenin is regulated by GSK-3beta, which is phosphorylated and downregulated by Akt. Endometrial carcinoma is the only tumor type, in which CyclinD1 mutations have been reported. Mutations of beta-catenin and Ras-PI3K pathway genes (such as K-Ras, PTEN and PIK3CA) are also well-known in endometrial cancer. In this study, we analyzed alterations of Cyclin D1 and beta-catenin, and addressed the relationship between these alterations and the Ras-PI3K activating mutations in endometrial cancer.

Materials and methods: We extracted DNA from 91 primary endometrial carcinomas (under informed consent) and 13 endometrial carcinoma cell lines. Mutations of CCND1 (Cyclin D1), CTNNB1 (beta-catenin), K-Ras, PTEN and PIK3CA were analyzed by PCR-direct sequencing. In 24 cliniclal samples, Single Nucleotide Polymorphism (SNP) typing arrays (50K) were performed to evaluate copy number alterations in 11q13.3 for CCND1, 3p22.1 for CTNNB1, 12p12.1 for K-Ras, 10q23.3 for PTEN and 3q26.3 for PIK3CA.

Result: Alterations of Cyclin D1 were detected in three clinical samples; mutations in two (2%) and amplification in one (4%) samples. Mutations of beta-catenin were detected in nine clinical samples (10%) and four cell lines (31%). No overlapping alterations were observed in Cyclin D1 and beta catenin. All the tumors with alterations in Cyclin D1 or beta-catenin coexist with mutations in the Ras-PI3K pathway genes (K-Ras, PTEN and PIK3CA). All the three patients with Cyclin D1 alterations were poor prognostic, whereas all the nine patients with beta-catenin mutations were alive without recurrence.

Conclusion: Our data suggest that tumors with mutations in beta-catenin or Cyclin D1 could more efficiently accelerate cell cycle by cooperating with the coexistent alterations in the Ras-PI3K pathway genes. Furthermore, Cyclin D1 mutation might be associated with tumor aggressiveness and poor prognosis in endometrial cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2957. doi:10.1158/1538-7445.AM2011-2957
Abstract 2959: The Syk tyrosine kinase negatively affects cell cycle progression through phosphorylation of the Cdk1 kinase in response to DNA damage
Authors: Saen Michael , Morel Anne , Larive Romain , Urbach Serge , Jouin Patrick , Morris May C. , Dulic Vjekoslav , Mangeat Paul , Coopman Peter J.
Published: June 2012

The non-receptor spleen tyrosine kinase Syk has mainly been studied in hematopoietic cells in which it plays a key role in the immune-response signalling. We previously demonstrated that Syk is also present in mammary epithelial cells and that its expression is lost during malignant progression of breast cancer cell lines. Clinical studies confirmed loss of Syk expression, not only in breast tumors but also in other carcinomas and melanoma. Xenotransplantation animal experiments evidenced that Syk acts as a tumor and metastasis suppressor in breast cancer and melanoma. The mechanisms by which Syk exerts its anti-oncogenic activity remain, however, unrevealed. We previously demonstrated that Syk localizes to the centrosomes and negatively affects cell division. The centrosomal Syk substrates and possible effectors of its anti-oncogenic activity remain, nevertheless, unknown. In this study, we demonstrate that Syk may negatively affect cell cycle progression through the phosphorylation of the Cdk1 kinase, a key protein involved in the control of mitosis.

Using immunofluorescence, we observed that endogenous Syk and Cdk1 colocalize at the centrosomes and that transient DsRed-Syk transfection induces pY15-Cdk1-positive epitopes at the centrosomes. In vitro kinase assays demonstrate that Syk can phosphorylate Cdk1 in the presence of different cyclins. Mass spectrometry allowed identification of four different Cdk1 tyrosine residues phosphorylated by Syk, amongst which the Y15 is known to negatively affect mitotic entry when phosphorylated by the Wee1 kinase. Transient overexpression of DsRed-Syk or GFP-Cdk1 carrying phospho-mimicking mutations resulted in a cell cycle block as observed by FACS analysis and led to a senescence-like growth arrest. Finally, we demonstrate that DNA damage-inducing agents induce Syk activation and Cdk1 tyrosine phosphorylation in a Syk-dependent manner, as evidenced by the use of a Syk-targeting shRNA.

In conclusion, our results reveal that Syk overexpression or Syk activation by genotoxic agents induce a cell cycle arrest through the phosphorylation of Cdk1. These novel aspects of Syk function will contribute to a better understanding of its onco-suppressive activity and, more generally, of the DNA damage-induced signalling.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2959. doi:10.1158/1538-7445.AM2011-2959
Abstract 2961: Spatiotemporal regulation of the SCF ubiquitin ligase component, Fbxo7
Authors: Nelson David E. , Laman Heike
Published: June 2012

Fbxo7 is a member of the F-box family of proteins which direct the E3 ubiquitin ligase activity of the SCF holoenzyme. Apart from the SCF, Fbxo7 is also able to function as an assembly scaffold for Cyclin D/Cdk6 complexes, promoting cellular transformation. The expression of Fbxo7 in immortalised embryonic fibroblasts rendered them invasive and capable of anchorage independent growth and tumour formation in nude mice. Moreover, transformation was dependent on the presence of Cdk6. In recent experiments we showed that the over-expression of Fbxo7 in haematopoietic stem cells cooperated with p53 mutation to promote T cell lymphoma in adoptive transfer experiments conducted in mice, demonstrating it is a sensitive and causal agent in oncogenesis. In human cancers, Fbxo7 was found to be highly expressed in various tumours, including haematopoietic malignancies, as well as lung and colon carcinomas. Interestingly, in a number of these tumour samples, the subcellular distribution of Fbxo7 was altered, changed from predominantly cytoplasmic to nuclear, suggesting that the localization of Fbxo7 is critical to its function.

We found that in proliferating cells, the localization of Fbxo7 was altered during the cell cycle phase: cytoplasmic during G1, and accumulating in the nucleus during S-G2. We have identified a functional NES motif embedded within the F-box domain of Fbxo7, the region which regulates its interaction with the SCF holoenzyme through binding to Skp1. Loss of the F-box domain it its entirety or targeted mutation of the NES renders the protein constitutively nuclear. We find also that binding of Fbxo7 to Skp1 prevents its interaction with Crm1, a component of the nuclear export machinery suggesting that association with the other SCF components directly promotes the nuclear accumulation of the protein. These findings suggest that during the latter part of the cell cycle SCF(Fbxo7) ubiquitin ligase activity is directed against a nuclear protein. A recent proteomics screen has now revealed new substrates for Fbxo7 within the nucleus which may help to decipher how the deregulation of the spatiotemporal regulation of Fbxo7 contributes to its role in cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2961. doi:10.1158/1538-7445.AM2011-2961
Abstract 2962: A rapid alternative method for cell cycle analysis using the Cellometer Vision
Authors: Chan Leo L. , Zhong Xuemei , Qiu Jean , Li Peter , Lin Bo
Published: June 2012

Traditional cell cycle analysis instrumentation involves the use of fluorescence microscopy, laser scanning cytometry, and flow cytometry. In this work, we demonstrate a rapid alternative device, the Cellometer Vision, which utilizes an imaging cytometry method for analyzing cell cycle measurement. This method employs both bright-field and fluorescence imaging of a disposable cell counting chamber to quickly provide concentration and percentages of cell subpopulations. Signal measurement and data analysis could be performed less than five min. Cell cycle assay was demonstrated by detection of changes in phase percentage within a cell population when Jurkat cells were induced with three different drugs, nocodazole, etoposide, and aphidicolin that arrest cell cycle at G2/M, S, and G0/G1 phase, respectively. Jurkat cells were fixed with 75% ethanol and stained with propidium iodide. Individual cells were identified by the software and the fluorescence intensity was measured from within the cell. The imaging cytometry method was able to detect and analyze fluorescence from the Jurkat cells, which generated cell cycle data plot for each drug at three different concentrations. The cell cycle analysis results were highly consistent with the results obtained from FACScalibur flow cytometer. This imaging cytometry method provides a rapid and cost-effective tool for cell concentration measurements, as well as cell cycle analysis, which may benefit smaller research laboratories, where flow cytometer is not readily available.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2962. doi:10.1158/1538-7445.AM2011-2962
Abstract 2968: The functional relationship between CHFR and PARP-1 controls the antephase checkpoint and tumor development
Authors: Kashima Lisa , Mita Hiroaki , Idogawa Masashi , Toyota Minoru , Sasaki Yasushi , Tokino Takashi
Published: June 2012

The mitotic checkpoint gene CHFR is silenced by promoter hypermethylation or mutated in various human cancers, suggesting that CHFR is an important tumor suppressor. Recent studies have reported that CHFR functions as an E3 ubiquitin ligase of Aurora A and HDAC1, resulting in the degradation of those proteins. To better understand how CHFR suppresses cell cycle progression and tumorigenesis, we sought to identify CHFR-interacting proteins using affinity purification combined with mass spectrometry. Poly (ADP-ribose) polymerase-1 (PARP-1), which catalyzes polyADP-ribosylation, was newly identified as a CHFR interacting protein. CHFR promoted polyubiquitination of PARP-1. Conversely, PARP-1 poly(ADP-ribosyl)ated CHFR. In addition, we mapped the region of both CHFR and PARP-1 required for the interaction. Because CHFR delays entry into metaphase in response to mitotic stress, we investigated whether PARP-1 is involved in the mitotic checkpoint. In the presence of PARP-1, CHFR arrested the cell cycle. On the other hand, in the absence of PARP-1, CHFR had no effect on the cell cycle arrest. Additionally, we found the knockdown of PARP-1 resulted in cell cycle arrest itself. As overexpression of PARP-1 has been reported in several human tumors, our findings suggest the functional relationships between CHFR and PARP-1 could play an important role in regulation of cell cycle and tumor suppression.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2968. doi:10.1158/1538-7445.AM2011-2968
Abstract 2971: The modulation checkpoint signaling by natural products during the DNA damage response
Authors: Nishida Hiroshi , Kawakami Kayoko , Tatewaki Naoto , Hirayama Masao , Ikekawa Nobuo , Konishi Tetsuya
Published: June 2012

ATM (ataxia telangiectasia mutated) and ATR (AT and Rad3 related) protein kinases play a crucial role in cellular DNA damage responses. The inhibition of these kinases and/or related signaling molecules lead to an abolishment of one such signal pathways termed as checkpoints. It is expected that the discovery of checkpoint modulator such as natural products will be an effective assistance of anti-cancer therapy with lower side effect. In the present study, we demonstrated the inhibitory effect of Squalene (SQ), flavonoids derived from Persimmon leaves (PL) and Schisandrin-B (SchB) on ATM/ATR-dependent checkpoint signaling pathway. The cell viability was decreased by SQ, PL or SchB treated A549 adenocarcinoma after gamma-irradiation (IR) or UV exposure. In each SQ, PL and SchB treated cells, G2/M checkpoint activity was abolished in DNA damaged cells. In vitro kinase assay revealed that PL and SchB inhibits ATM and ATR, respectively. SQ, on the other hand, showed no effect on both kinases. Interestingly, SQ increased Wip1 protein expression in unperturbed cells, and suppressed ATM activation in IR treated cells. Consistent with potential inhibition of ATM by SQ or PL, IR-induced phosphorylations of ATM effectors such as p53 (S15) and Chk1 (S317) were remarkably inhibited by the treatment of SQ or PL. SchB inhibited the specific phosphorylation of these effectors by ATR. Moreover, Xenograft and allograft studies with natural products were demonstrated that the tumor growth of both cancer cells in vivo were obviously inhibited by SQ, PL or SchB treatment. These results suggest that some natural products such as SQ, PL and SchB might be applicable to the sensitization of anti-cancer therapies such as radio- /chemotherapy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2971. doi:10.1158/1538-7445.AM2011-2971
Abstract 2973: Differential regulation of p21waf1 protein half-life by DNA damage and nutlin-3 in p53 wild-type tumors and its therapeutic potential
Authors: Chang Li-Ju , Eastman Alan R.
Published: June 2012

DNA damage induces cell cycle arrest through both Chk1 and the p53 tumor suppressor protein, the latter arresting cells through induction of p21waf1 protein. Arrest permits cells to repair the damage and recover. The frequent loss of p53 in tumor cells makes them more dependent on Chk1 for arrest and survival. We have used the topoisomerase I inhibitor SN38 to induce DNA damage in three p53 wild-type cell lines: the immortalized MCF10A, and tumorigenic HCT116 and U2OS. Both phospho-Chk1 and p53 were elevated to a similar extent in all three cell lines. However, the HCT116 and U2OS cells exhibited far less accumulation of p21waf1 protein. The p21waf1 mRNA was induced to a similar extent, and the protein half-life was similar (∼ 30 min) in all three cell lines. These results suggest that the two tumor cell lines have a reduced rate of translation of the p21waf1 mRNA. To obtain further insight into the regulation of p21waf1, we incubated the three cell lines with the non-genotoxic drug nutlin-3, which disrupts p53:MDM2 binding thereby inducing a p53 response. Nutlin-3 induced similar levels of p53 in the three cell lines, and these levels were comparable to those seen with SN38. While nutlin-3 had little additional impact on p21waf1 induction in MCF10A cells and the half-life remained at ∼ 30 min, there was a much stronger induction in HCT116 and U2OS cells which was attributed to a much longer protein half-life (∼ 80 to 90 min). Incubation of p53 mutant cells with Chk1 inhibitors such as SCH900776 can overcome SN38-induced cell cycle arrest and drive cells through a lethal mitosis. p53 wild-type cells can be protected from this lethal mitosis through induction of p21waf1. However, the defective p21waf1 induction in HCT116 and U2OS cells is insufficient to protect them from Chk1 inhibition. In contrast, incubation with nutlin-3 induced sufficient p21waf1 that the cells arrested in either G1 or G2. This arrest protected the cells from subsequent incubation with the S phase-specific DNA damaging agents SN38 and hydroxyurea, either alone, or when combined with checkpoint inhibition. This protection was not observed in MCF10A or HCT116 cells deleted for p21waf1, or in MDA-MB-231 cells that have mutant p53. The results demonstrate that some p53 wild-type tumors have defects in p21waf1 expression, and may retain sensitivity to a combination of DNA damage plus checkpoint inhibition. Furthermore, the results suggest nutlin-3 could be used to protect normal cells from these combinations such that therapy can be targeted selectively to p53-defective tumors.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2973. doi:10.1158/1538-7445.AM2011-2973
Abstract 2986: Partial inhibition of Plk1 is cytotoxic despite normal spindle structure
Authors: Lera Robert F. , Burkard Mark E.
Published: June 2012

Polo-like kinase 1 (Plk1) is a multifunctional mitotic kinase that has emerged as an attractive target for cancer chemotherapy. Frequently over-expressed in tumors, Plk1 is absent in the surrounding non-proliferating tissue. Furthermore, Plk1 depletion in tumor cell lines produces monopolar spindles and mitotic arrest that ultimately leads to cell death. However, it is unclear whether monopolar spindles mediate cytotoxicity at physiologic drug concentrations. We used a chemical genetic system to test the effect of partial inhibition of Plk1 in human cells. We discovered that partial inhibition of Plk1 produces a cytotoxic response in the absence of a prolonged mitotic delay. We hypothesized that partial inhibition disrupts mitosis via alternate mechanisms from the archetypal monopolar spindle-induced prometaphase arrest. We performed time-lapse microscopy experiments after exposure to low (0-1 μM) and high (10 μM) concentrations of the ATP analog, 3-MBPP1. As expected, 90% (53/60) of untreated cells successfully completed mitosis, whereas all cells (28/28) treated with 10 μM exhibited a monopolar-spindle induced prometaphase arrest. Surprisingly, 29-40% of cells treated with 3-MBPP1 exhibited a transient prometaphase delay, followed by impaired anaphase segregation and frequent cytokinesis failure. Indirect immunofluorescence demonstrates aberrations in chromosome and microtubule dynamics, which we are further characterizing. Partial inhibition of Plk1 leads to cytotoxicity by a spindle-independent mechanism. These results are important for developing Plk1-targeted therapies. First, they demonstrate that continuous dosing of an oral Plk1 inhibitor is likely to act by a different mechanism from intermittent high doses of an intravenous drug. Second, partial inhibition may provide a more physiologically relevant background to study function of kinase inhibitor activity in the treatment of cancer. Finally, identification of the responsible substrate may reveal the most sensitive biomarker for kinase inhibition in patient samples.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2986. doi:10.1158/1538-7445.AM2011-2986
Abstract 2989: Integrin-linked kinase regulates mitotic spindle dynamics and its overexpression leads to resistance to paclitaxel-induced cell death
Authors: Lim Simin , Fielding Andrew B. , Kawamura Eiko , Dedhar Shoukat
Published: June 2012

Dysregulated mitosis is a hallmark of cancer. Many centrosomal proteins, including the integrin-linked kinase (ILK), that regulate microtubule organization and mitotic spindle assembly have been implicated in cancers. Inhibition or depletion of ILK result in a mitotic spindle defect that has not been well characterized and while ILK is overexpressed in many cancers, the effect of ILK overexpression on mitotic spindle function is unknown. In this study, we have characterized the defective spindle phenotype, and also looked at the effect of ILK overexpression on mitotic spindle function. HeLa cells stably over-expressing wild-type ILK were used for overexpression studies while parental HeLa cells were treated with QLT-0267, an ILK-specific inhibitor, to study the defective spindle phenotype. While ILK inhibition led to an abnormal mitotic spindle phenotype, the spindle contained bona fide kinetochore-microtubules that persisted after cold- or calcium-treatment. ILK inhibition, however, led to higher sister-centromere stretch in chromosomes aligned between two spindle poles, suggesting that kinetochores were under abnormally high tension, likely due to inhibition of microtubule polymerization. Microtubule regrowth in mitotic cells after cold depolymerization was significantly slower with ILK inhibition. In the context of ILK overexpression in cancers, we first measured the duration of mitosis and showed that ILK-overexpressing cells completed mitosis at a faster rate and also required shorter durations between metaphase and anaphase. ILK overexpression also resulted in increased resistance to paclitaxel-induced cell death. Collectively, our data suggest that ILK regulates microtubule dynamics, which is the focus of ongoing investigations in our laboratory, and ILK overexpression has clinical implications by inducing resistance to paclitaxel, possibly through increased microtubule dynamics.

This work was supported by funding from the Canadian Institutes of Health Research.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2989. doi:10.1158/1538-7445.AM2011-2989
Abstract 2994: Constitutional telomere length correlates with colorectal cancer (CRC), but varies by age of cancer onset
Authors: Boardman Lisa A. , Johnson Ruth A. , Skinner Halcyon G. , Seo Songwon , Gangnon Ronald , Litzelman Kristin , Engelman Corinne , Thibodeau Stephen N. , Petersen Gloria M. , Cunningham Julie M.
Published: June 2012

Introduction: Short constitutional telomere length is associated with aging and cancer, but the correlation of telomere length and age at diagnosis of CRC has not been evaluated. Methods: We measured telomere length using quantitative PCR of peripheral blood leukocyte DNA in 1026 individuals diagnosed with MSS CRC who were ≤60 years of age and were chemo-radiotherapy naïve at the time of blood draw. We compared telomere length with 1972 unrelated healthy controls who were frequency matched for gender and age at time of blood collection. We used constructed percentiles of telomere length using the data from the controls. Results: Overall, regardless of cancer status, there was a weak negative correlation of telomere length with age such that as age increased telomere length decreased (Pearson correlation coefficient =−0.1, p< 0.0001); and men had shorter telomeres than women (p=0.0002). The odds ratio (OR) for MSS CRC increased with longer telomere length (OR 1.62 [CI: 1.31-1.99] for those with telomere lengths in the 90th percentile compared to those with telomere lengths in the 50th percentile. In multivariate analysis with adjustment for gender, BMI, smoking, alcohol, and diabetes, telomere length remained an independent risk factor for CRC (p< 0.001), with interaction between telomere length and age (≤50, longer telomeres increased CRC risk; > 50 years old, increasing telomere length associated with decreased CRC risk). Risk was higher for MSS CRC ≤50 years for patients with telomere lengths in the 90th percentile compared to those in the 50th percentile (OR 2.31; CI:1.65-3.23). For subjects diagnosed 50-60 yrs, longer telomere length was associated with lower risk of CRC, and shorter telomere length was associated with the development of CRC (OR 1.22 for telomere lengths in the 10th percentile compared to those in the 50th percentile (OR 1.22:CI; 1.05-1.43). Conclusion: Telomere dynamics appear to influence MSS CRC risk and may differ on the basis of age of cancer diagnosis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2994. doi:10.1158/1538-7445.AM2011-2994