bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1585: Loss of CDK5 sensitizes ovarian cancer cells to paclitaxel by enhancing intracellular retention
Authors: Ahmed Ahmed A. , Le Xiao-Feng , Lu Zhen , Mao Maggie , Aiken Abigail , Bast Robert C.
Published: February 2011

In order to improve the efficacy of paclitaxel in treatment of ovarian cancer, we have performed a kinome siRNA library screen to identify kinases that regulate paclitaxel sensitivity. CDK5 was one of kinase targets that could modulate paclitaxel sensitivity in SKOv3 cells. Unlike typical cyclin dependent kinases, CDK5 does not have a clearly defined role in the regulation of the cell cycle. CDK5 is required for the regulation of neuronal synaptic vesicular transport in the brain through phosphorylation of Munc 18, a key regulator of vesicular exocytosis. In this report, we aimed to explore the mechanisms by which CDK5 potentiate paclitaxel activity. We found that eighteen ovarian cancer cell lines expressed variable CDK5 levels measured by quantitative real time PCR and Western blotting. Basal levels of CDK5 in ovarian cancer cell lines appeared not to correlate with paclitaxel sensitivity. We used immunoprecipitation of CDK5 and in vitro kinase assays using 32P-ATP and observed that paclitaxel treatment of SKOv3 cells resulted in a profound (>20 fold) increase in CDK5 activity. Selective depletion of CDK5 using 4 individual siRNAs in SKOv3, TOV112 and DOV13 ovarian cancer cells lines resulted in a significant increase in paclitaxel-induced cytotoxicity as measured by cell viability assays. In addition, loss of CDK5 resulted in a significant increase in paclitaxel-mediated induction of caspase 3/7 activation in the ovarian cancer cell lines SKOv3, ES2, OVAR429 and DOV13. Remarkably, the magnitude of sensitization was directly proportional (p<0.001) to the dose of paclitaxel used. Knockdown of CDK5 did not enhance microtubule stabilization or increase paclitaxel-induced mitotic arrest. Instead, CDK5 depletion in SKOv3 cells resulted in a significant (p<0.001) increase in retention of fluorescent-tagged paclitaxel. This effect was similar to but independent from the effect of inhibition of trans-membrane pumps using verapamil, suggesting that the two mechanisms act in parallel. Indeed, knockdown of CDK5 in combination with verapamil resulted in greater intracellular retention of paclitaxel in SKOv3 cells. Consequently, we conclude that depletion of CDK5 results in decreased paclitaxel exocytosis leading to increased cytotoxicity to ovarian 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 1585.
Abstract 159: LINE-1 hypomethylation and patient prognosis in 1118 colorectal cancers
Authors: Ogino Shuji , Nosho Katsuhiko , Schernhammer Eva , Giovannucci Edward , Fuchs Charles
Published: February 2011

Background: Global DNA hypomethylation plays an important role in activation of oncogenes in a genome-wide scale, chromosomal instability and colorectal carcinogenesis. DNA methylation in LINE-1 (L1) repetitive element has been correlated with global DNA methylation level. We examined LINE-1 methylation in colorectal cancer and patient survival.

Methods: Utilizing 1118 colorectal cancers in two prospective cohorts, we quantified methylation in a LINE-1 by Pyrosequencing. Kaplan-Meier method and Cox proportional hazard models were used to assess differences in colon cancer-specific and overall mortalities in univariate analysis and multivariate analysis that adjusted for patient characteristics and tumoral molecular features, including BRAF mutation, the CpG island methylator phenotype and microsatellite instability.

Results: LINE-1 hypomethylation in 875 colon cancers was independently and linearly associated with high colon-cancer specific mortality (Ptrend<0.0001) and overall mortality (Ptrend=0.0004). Compared to patients with ≥75% LINE-1 methylated tumors, the adjusted hazard ratio (HR) for colon cancer-specific mortality were 1.36 [95% confidence interval (CI), 0.76-2.41] for 60-75% LINE-1 methylated tumors, 1.98 (95% CI, 1.10-3.55) for 45-60% LINE-1 methylated tumors, and 3.83 (95% CI, 1.87-7.84) for <45% LINE-1 methylated tumors. Similar findings were noted for overall mortality. Kaplan-Meier analysis demonstrated shorter survival among patients with LINE-1 hypomethylated tumors (log-rank p<0.0001). The prognostic influence of LINE-1 hypomethylation in colon cancer was consistent across the two independent cohort studies as well as across strata of clinical or molecular characteristics. In contrast, LINE-1 hypomethylation was not significantly associated with survival of 243 rectal cancer patients (adjust HR 1.02; 95% CI, 0.48-2.19; for a 30% decrease of LINE-1 methylation; Ptrend=0.96).

Conclusions: Tumoral LINE-1 hypomethylation is associated with shorter survival among colon cancer patients.

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 159.
Abstract 1595: Phosphomimetic mutation of hnRNP A1 inhibits AKT-dependent IRES function and results in mTOR inhibitor sensitivity
Authors: Martin Jheralyn , Masri Janine , Cloninger Cheri , Holmes Brent , Lichtenstein Alan , Gera Joseph F.
Published: February 2011

The relative activity of the Akt kinase has been demonstrated to be a major determinant of sensitivity of tumor cells to mTOR inhibitors. Our previous studies have shown that the multifunctional RNA-binding protein hnRNP A1 regulates a salvage pathway which facilitates IRES-dependent mRNA translation of critical cellular determinants in an Akt-dependent manner following mTOR inhibitor exposure. This salvage pathway functions by stimulating IRES-dependent translation in cells with relatively quiescent Akt resulting in resistance to rapamycin. However, the pathway is repressed in cells with elevated Akt activity rendering cells sensitive to rapamycin induced G1 arrest as a result of the inhibition of global eIF-4E-mediated translation. Akt phosphorylation of hnRNP A1 at serine 199 has been demonstrated to inhibit IRES-mediated translation initiation. Here we describe a phosphomimetic mutant of hnRNP A1 (S199E) which binds both the cyclin D1 and c-MYC IRES RNAs in vitro and results in inhibition of IRES function in dicistronic mRNA reporter assays. Utilizing cells in which Akt is conditionally active, we demonstrate that overexpression of this mutant renders quiescent Akt-containing cells sensitive to rapamycin in vitro and in xenografts. We also demonstrate that activated Akt is strongly correlated with elevated phospho- ser199-hnRNP A1 levels in a panel of 22 glioblastomas. These data demonstrate that the phosphorylation status of hnRNP A1 ser199 regulates the Akt-dependent sensitivity of cells to rapamycin and may have utility as a surrogate biomarker to stratify those tumors which may be most sensitive to mTOR inhibitor therapy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1595.
Abstract 1597: Expression of caPCNA and antineoplastic action of caPeptides in pancreatic cancer cells
Authors: Shen Fei , Hickey Robert , Kamran Jasmine , Phipps Elizabeth A. , Malkas Linda H.
Published: February 2011

Our laboratory previously reported the identification of an acidic isoform of proliferating cell nuclear antigen (caPCNA) in various cancer cells, which appears to be associated, at least in part, with malignant transformation of cells. The current studies show the expression of caPCNA in BXPC-3, Paca-2 and Capan-1 pancreatic cancer cells. Since Proliferating Cell Nuclear Antigen (PCNA) is involved in DNA replication and repair in prokaryote and eukaryote cells, we hypothesize that caPCNA is likely to perform similar functions specifically in cancer cells. Antibodies developed against caPCNA showed growth inhibition activity in cancer cells suggesting caPCNA is related to the proliferation of malignant cells. In order to begin to understand the function of caPCNA in cellular malignant transformation, we have investigated the interaction of caPCNA with its binding partners including flap structure-specific endonuclease 1(Fen-1) and xeroderma pimentosum complementation group G (XPG). We have identified an amino acid domain sequence that confers the cancer cell specificity of caPCNA. In the course of our studies we synthesized small caPCNA related peptides (caPeptides) derived from this sequence that promote cancer cell cytotoxicity. caPeptides have the potential ability to compete with several cellular proteins that participate in DNA replication, repair, cell cycle control, transcription, and chromosomal recombination for their naturally occurring binding site on caPCNA and block binding of full length caPCNA to these proteins. The disruption of the interaction between caPCNA and its binding proteins prevents cancer cellular functions (vital cellular machinery) that recruit caPCNA and therefore may lead caPCNA-derived peptides to be cytotoxic by themselves or in combination with cancer chemotherapeutic drugs. These peptides, either alone or in combination with other anticancer agents are potentially useful cancer chemotherapeutics or augmentors of the pharmacodynamic effect of specific anti-cancer chemotherapeutics. Our results show that these peptides demonstrate promising growth inhibition and cytotoxicity in pancreatic cancer cells. Considering that pancreatic cancer is well known for its high mortality rate due to its chemotherapy ineffectiveness our studies render further investigations on the action of caPCNA and the anti-neoplastic mechanism of the peptides in pancreatic 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 1597.
Abstract 1599: Expression of therapeutics targets in osteosarcoma : Effects of chemotherapy and evolution of disease
Authors: Versleijen-Jonkers Yvonne M.H. , van de Luijtgaarden Addy C.M. , Leus Manon A. , Roeffen Melissa H.S. , Flucke Uta E. , van der Geest Ingrid C.M. , van der Graaf Winette T.A.
Published: February 2011

Introduction

The receptor activator of nuclear factor kb/-ligand/osteoprotegerin (RANK/RANKL/OPG) triad and insulin-like growth factor 1 receptor (IGF1R) system are potential therapeutic targets in osteosarcoma. We studied the consistency of expression of specific targets over time and across lesions within subjects. This aids to select biomarkers for future therapies and to determine an optimal sequence of targeted and cytotoxic therapy.

Materials and methods

Immunohistochemical staining was performed on paraffin-embedded therapy-naïve biopsies, post-neoadjuvant chemotherapy resection specimens, and metastatic lesions for RANK, OPG, IGF1R, IGF1, phosphorylated protein kinase B (pAKT), mammalian target of rapamycin (pMTOR) and extracellular-regulated kinase (pERK). Staining intensity was scored as absent (0), weak (1) or strong (2). Change of expression between lesions within subjects was interrogated by Wilcoxon signed rank tests.

Results

49 tumor samples of 28 patients were available (staining results presented in table 1). No change in RANK or OPG expression was observed. After chemotherapy pERK (p=0.008) and pMTOR expression (p=0.015) decreased; pERK and IGF1R expression (both p=0.046) were lower in metastases than in primary tumors. Expression of IGF1 and pAKT remained unchanged.

Discussion

The choice for a therapeutic regimen is increasingly guided by presence of specific molecular alterations in tumor tissue. Variation of target expression over time and across lesions within subjects is a known phenomenon in common cancers. In osteosarcomas, we found a rather consistent expression signature with the exception of pERK, pMTOR and IGF1R, which were downregulated after exposure to chemotherapy or in metastatic lesions.

Conclusion

Screening for biomarkers for specific targeted drugs in osteosarcoma should not be limited to a single tumor sample. It is likely from our data that an optimal sequence of targeted therapies and cytotoxic drugs exists.Table 1:Staining results (%) Biopsy (n=28)Resection (n=13)Metastasis (n=8)Intensity012012012RANK014860158513088OPG4544383954255025IGF140960217900100IGF1R778681577145729pmTOR252946691515431443pAKT11503931393129710pERK001005404650050

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 1599.
Abstract 1603: The VEGF, PDGF and STAT3 pathways are the potential therapeutic targets to treat endometrial cancer
Authors: Korovkina Victoria P.
Published: February 2011

Progesterone inhibits growth of endometrial cancer through its receptors A (PRA) and B (PRB) although the molecular mechanisms of inhibition have yet to be fully characterized. A global analysis of gene regulation in human endometrial cancer cells that express heterologous progesterone receptors was performed to identify the candidate genes that mediate inhibitory effects of progesterone in endometrium. Data indicate that progesterone down-regulates the expression of vascular endothelial growth factor A (VEGFA), platelet derived growth factor A (PDGFA) and signal transducer and activator of transcription 3 (STAT3). Down-regulation of VGFA is mediated by the PRA whereas both PRA and PRB are required to attenuate expression of PDGFA and STAT3 genes. These results suggest that progesterone exerts the growth-limiting effects in endometrium via inhibition of angiogenesis and proliferation. VGFA, PDGFA and STAT3 pathways may be central to the inhibitory effects of progesterone in endometrial cancer and may constitute the important targets for therapeutic interventions.

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 1603.
Abstract 1604: HFE is a new target molecule for cancer treatment
Authors: Lee Sang Y. , Slagle-Webb Becky , Sheehan Jonas M. , Connor James R.
Published: February 2011

Iron can enhance cell growth and metabolism. However insufficiently regulated iron can lead iron accumulation, enhancing oxidative stress and subsequent damage to DNA, and finally promote cancer. The HFE protein plays a key role in the regulation of body iron and is part of the innate immune system. HFE is the most commonly occurring polymorphism in Caucasians. The two most common HFE polymorphic alleles are C282Y and H63D. The variant forms of HFE have increased iron accumulation. To see the impact of HFE on the brain tumor development, at first, we determined the HFE expression. HFE strongly express in the brain tumors such as meningioma, glioblastoma, and astrocytoma at immunohistochemistry. Then, to assess the association between HFE variants and growth, we measured cell growth and tumor development. We found that human neuroblastoma cell lines that were stably transfected with the C282Y allelic HFE variant proliferated at a much higher rate than the same cell lines expressing the wild type or H63D gene variant at the cell proliferation assay and counting cell number. Furthermore, nude mice injected with glioma cells with the C282Y allele developed much faster and bigger tumor than the other type of HFE cell lines. Next, we transfected HFE siRNA into the cells to determine if the increased rate of proliferation was directly related to the protein associated with the C282Y allelic variant. HFE siRNA treatment significantly inhibited cell proliferation of C282Y transfected neuroblastoma cells. The effect was dose dependent. The cell growth was over 60 % decreased for cells cultured for 4 days after exposure to 15 nM of HFE siRNA. Meanwhile, cells transfected with non-specific siRNA (negative control) or the transfection reagent (mock) control siRNA showed no evidence of inhibited cell proliferation. HFE siRNA also inhibited cell proliferation of wild type and H63D allele cells. We also observed that HFE siRNA inhibited tumor development in the glioma cells injected nude mice. We are currently studying specific delivery system of HFE siRNA to the tumor cells. In summary, we found that HFE inhibition is enough to prevent cancer cell proliferation and tumor development.

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 1604.
Abstract 1609: Functional analysis of SFRS10: A novel molecular target for anti-cancer drug
Authors: Wada Satoru , Eguchi Hidetaka , Tanimoto Keiji , Hiyama Keiko , Nishiyama Masahiko
Published: February 2011

[Background]

Development of novel molecular-target drugs is actively conducted: A large number of such drugs are under clinical trials. However, an ideal molecular-target drug with high-specificity and low-toxicity is yet to be developed. For epoch-making development of cancer drugs, it is necessary to further identify target candidates by unprecedented approach.

[Purpose]

Last year, we reported 13 candidate genes that contributed to an immortalization through comprehensive gene expression analysis of human mortal- and immortal-cells and tissues. Under the same procedure, we here added SFRS10 gene to the list. To evaluate its possibility as drug target, we attempted here a functional analysis of SFRS10.

[Materials and Methods]

Human colon cancer cell lines DLD-1 and HCC48 and pancreatic cancer cells MIAPaCa-2, KLM-1, and BxPC-3 were used in each experiment. Quantitative analyses of gene expression levels were conducted by real-time RT-PCR method using gene specific-primers and Universal Probe Library probes (Roche). Microarray analysis was conducted with 4×44K Whole Human Genome Microarray (Agilent). Pathway analysis was performed through the use of Ingenuity Pathways Analysis (IPA, Ingenuity® Systems, www.ingenuity.com). Knockdown experiments were performed using Stealth RNAi and RNAiMax reagent (Invitrogen). Cell proliferation was measured with MTT assay. Caspase activities were analyzed using ApoAlert Caspase Profiling Plate (Clontech). Cell cycle profile was obtained by measuring PI-stained cells with FACS “Calibur” (BD Biosciences).

[Results]

SFRS10 knockdown induced inhibition of cell proliferation of all the tested cells. Because knockdown efficiencies and cytostatic activities showed a good correlation in the experiment, we speculated that SFRS10 may have an important role for the cell proliferation. Treatment with SFRS10 siRNA induced cell cycle arrest in the S and G2/M phases and apoptosis in DLD-1 and KLM-1 cells. Moreover, we observed that some cell cycle-related genes, such as MAD2L1 and RFC4, altered their expression levels in response to SFRS10 knockdown as assessed by microarray analysis; cell cycle checkpoint pathways were significantly changed with network analyses. The alteration of gene expression levels were confirmed by real-time RT-PCR analyses.

[Conclusion]

These results suggested that SFRS10 may have an important role in regulation of cell proliferation and may control cell cycle checkpoints. Taken together, SFRS10 may be one molecule responsible for immortalization of cancer cells and the useful molecular target for cancer therapy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1609.
Abstract 161: Discovery of novel methylation-based biomarkers for epithelial ovarian cancer using oligonucleotide microarrays
Authors: de Graeff Pauline , Wisman Bea , Fehrmann Rudolf SN , Crijns Anne PG , Meersma Gert Jan , Volders Haukeline H. , Blijdorp Iris , Mourits Marian JE , Hollema Harry , de Jong Steven , van der Zee Ate GJ
Published: February 2011

Objective: To improve current screening modalities, additional molecular markers that allow detection of ovarian cancer at an early stage are needed. The aim of the current study was to identify novel methylation-based biomarkers for ovarian cancer.

Methods: Genes frequently expressed at low levels were identified by comparing global expression levels from 232 primary ovarian cancers to Universal Reference RNA. Ten genes possessing a CpG island in the promoter region that showed frequent low expression in serous cancers and at least one other histological subtype were selected. The methylation status of candidate genes was verified in 50 sporadic ovarian cancers, 11 hereditary cancers, 13 borderline cancers and 12 cystadenomas using methylation specific PCR (MSP).

Results: Three candidate genes (IGFBP1, LIN28 and ZNF582) showed frequent methylation in cancers and were unmethylated in normal leukocyte DNA and human ovarian surface epithelial cells. Promoter methylation of any of the three candidate genes was observed in 88% of stage III/IV cancers and 72% of stage I/II cancers. In contrast, only 9% of hereditary cancers showed evidence of methylation for any of the three genes (p<0.001). IGFBP1 was mainly methylated in stage III/IV cancers (88% vs. 12%, p<0.001), while methylation of LIN28 and ZNF582 was more frequent in stage I/II cancers (8% vs. 44% and 8% vs. 48%, respectively; p<0.05).

Conclusions: Using oligonucleotide microarray data, three novel methylation-based biomarkers for sporadic epithelial ovarian cancer were discovered. Further studies should elucidate the methylation status of these genes in larger cohorts of ovarian cancers and investigate their methylation status in serum.

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 161.
Abstract 1612: Validation of CSE1L/hCAS as a potential therapeutic target in pancreatic cancer
Authors: Husain Kazim , Coppola Domenico , Centeno Barbara , Helm James , Kooman John , Lawrence Nicholas , Sebti Said , Malafa Mokenge
Published: February 2011

Background: The nuclear export protein CSE1L/hCAS has recently gained attention as a putative oncogene, which is amplified in several human malignancies. The role of hCAS in pancreatic cancer is unknown. In this work, we sought to evaluate the expression of hCAS in pancreatic cancer cell lines and tumor tissue and to further explore the potential of hCAS as a therapeutic target.

Methods: The nuclear and cytosolic hCAS protein expression was determined in human pancreatic cancer cell lines and tumor tissues and compared with those of immortalized normal pancreatic ductal cells and normal pancreatic tissues. The cellular effects of hCAS knockdown using small interfering RNA oligonucleotides were investigated in MiaPaCa-2 human pancreatic cancer cells with regard to proliferation (MTT assay), cell death (Trypan blue and Flow cytometry), and malignant transformation (colony formation assay).

Results: hCAS protein expression increased in pancreatic cancer cell lines relative to the immortalized pancreatic ductal epithelial cell line. Immunohistochemical staining of a human pancreatic tumor tissue microarray showed that hCAS expression was higher in pancreatic tumors compared with their normal counterparts. There was increasing nuclear to cytoplasmic staining with progression from normal pancreatic duct to increasing grade of pancreatic intraductal neoplasia to invasive pancreatic cancer. Treatment with hCAS targeting small interfering RNAs effectively reduced pancreatic cell growth in tissue culture, induced apoptosis, and inhibited growth in soft agar.

Conclusion: This is the first report of hCAS expression and cellular distribution in human pancreatic cancer. Our data suggest that hCAS might be an attractive target for human pancreatic cancer prevention and therapy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1612.
Abstract 1617: Potential therapeutic effect of glycogen synthase kinase 3β inhibition against human glioblastoma
Authors: Miyashita Katsuyoshi , Kawakami Kazuyki , Nakada Mitsutoshi , Shakoori Abbas , Fujisawa Hironori , Hayashi Yutaka , Hamada Jun-ichiro , Minamoto Toshinari
Published: February 2011

Purpose: Glioblastoma represents the malignant brain tumor that is most refractory to treatment and in which the identification of molecular target(s) is urgently required. We investigated the expression, activity, and putative pathologic role of glycogen synthase kinase 3β (GSK3β), an emerging therapeutic target for neurodegenerative diseases, in human glioblastoma.

Experimental Design: The active fractionof GSK3β that is phosphorylated at the tyrosine 216 residue (pGSK3βY216) was identified in glioblastoma cell lines. GSK3β activity for phosphorylating its substrate was detected in these cells by nonradioisotopic in vitro kinase assay.

Results: Higher expression levels of GSK3β and pGSK3βY216 were frequently detected in glioblastomas compared with nonneoplastic brain tissues. Inhibition of GSK3β activity by escalating doses of a small-molecule inhibitor (AR-A014418) or inhibition of its expression by RNA interference induced the apoptosis and attenuated the survival and proliferation of glioblastoma cells in vitro. Inhibition of GSK3β was associated with increased expression of p53 and p21 in glioblastoma cells with wild-type p53 and with decreased Rb phosphorylation and expression of cyclin-dependent kinase 6 in all glioblastoma cell lines. Administration of AR-A014418 at a low dose significantly sensitized glioblastoma cells to temozolomide and 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea, chemotherapeutic agents used in the clinical setting, as well as to ionizing radiation.

Conclusion: These results indicate thatGSK3β exerts a pathologic role by promoting the survival and proliferation of glioblastoma cells and by protecting them from apoptosis via the inactivation of p53- and/or Rb-mediated pathways (Clin Cancer Res 2009;15:887-97). Consequently, we propose that GSK3β provides a potential therapeutic target in glioblastoma (Minamoto T, inventor; National University Corporation Kanazawa University; assignee. Suppression of cancer and method for evaluating anticancer agent based on the effect of inhibiting GSK3β. International patent WO2006/073202. 2006 Jul 13. United States patent US 11/794,716. 2006 Jan 4. European patent EP1845094 2007 Oct 17. Japan patent 2006-550915 2007 Jun 21).

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 1617.
Abstract 162: DNA methylome in familial breast cancer identifies distinct profiles defined by mutation status
Authors: Flanagan James , Cocciardi Sibylle , Waddell Nic , Johnstone Cameron , Henderson Stephen , Simpson Peter , da Silva kConFab, Leonard , Khanna Kumkum , Lakhani Sunil , Boshoff Chris , Chenevix-Trench Georgia
Published: February 2011

It is now understood that epigenetic alterations occur frequently in sporadic breast carcinogenesis, but little is known about the epigenetic alterations associated with familial breast tumors. We performed genome-wide DNA methylation profiling on frozen, familial breast cancers (n=33) to identify patterns of methylation specific to the different mutation groups (BRCA1, BRCA2 and BRCAx) or intrinsic subtypes of breast cancer (basal, luminal A, luminal B, HER2-amplified and normal-like). We used methylated DNA immunoprecipitation (meDIP) on Affymetrix promoter chips to interrogate methylation profiles across 25,500 distinct transcripts, and performed technical validation of multiple loci with Epityper. Using a support vector machine classification algorithm, we demonstrated that genome-wide methylation profiles predicted tumor mutation status with estimated error rates of 19% (BRCA1), 31% (BRCA2) and 36% (BRCAx), but did not accurately predict the intrinsic subtypes defined by gene expression with error rates of 43% (basal) and 54% (luminal A). Furthermore, using unsupervised hierarchical clustering we identified a distinct subgroup of BRCAx tumors defined by methylation profiles. We then validated these findings in the original 33 tumours and in an independent set of 47 formalin-fixed, paraffin embedded BRCA1 (n=14), BRCA2 (n=13) and BRCAx (n=20) tumours by Pyrosequencing. “Finally, gene expression profiling and SNP CGH array previously performed on the same samples allowed full integration of methylation, gene expression and copy number data sets. This integrated analysis revealed frequent hypermethylation of genes that also displayed loss of heterozygosity (LOH) compared to the tumors that were diploid for that gene. We also observed frequent hypermethylation of genes that show copy number gains compared to diploid tumors providing a potential mechanism for expression dosage compensation. Together these data show that methylation profiles for familial breast cancers are defined by the mutation status and distinct from the intrinsic subtypes.

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 162.
Abstract 1625: Discovery of a breast cancer targeting peptide with anti-tumor activity
Authors: Proneth Bettina , Driessen Wouter H. , Hajitou Amin , Staquicini Fernanda I. , Arap Wadih , Pasqualini Renata
Published: February 2011

The American Cancer Society estimates 192,370 women will be diagnosed with breast cancer in the United States in 2009, and approximately 40,170 will die of this disease. Towards identifying new ligand-directed therapies for breast cancer, we have developed an in vivo screening method, where peptides that home specifically to the vascular component of tissues are selected after intravenous administration of an engineered combinatorial peptide phage library. We have used this technology for the identification of a new breast cancer targeting peptide and its receptor in a mouse model of highly aggressive and vascularized breast cancer.

A random phage library displaying the cyclic sequence CX7C (X = any of the 20 natural amino acids) was injected intravenously in tumor-bearing mice after which phage was recovered from the tumor-tissue by bacterial infection and re-injected for a subsequent round of biopanning. After sequencing of the recovered phage from round 2 and 3 several candidate peptides were identified. One breast cancer targeting peptide (BCTP) was recovered from all tumors with the highest frequency. This peptide showed marked tumor homing as verified by immunohistochemistry (IHC). We used peptide affinity chromatography and tandem mass spectrometry (MS/MS) to identify the target receptor of BCTP. Strikingly, this newly discovered receptor plays an as yet unrecognized role in the pathology of breast cancer. BCPT exhibited strong binding to its receptor as verified by ELISA. To investigate if the BCTP receptor is a relevant candidate drug target in breast cancer, we obtained biopsy samples of breast cancer patients prior to chemotherapy. Strong immunoreactivity of the BCTP receptor was observed in the paraffin embedded tissue sections. Next, mammary tumor-bearing mice were injected twice per week with BCTP, control peptide or saline, systemically. BCTP treated mice demonstrated a marked decrease in tumor size of treated mice, compared to mice injected with PBS alone.

We have identified, characterized and validated the peptide BCTP, specifically homing to highly aggressive, vascularized breast tumors. In addition to its tumor targeting capabilities this peptide possesses anti-tumor function in itself, inhibiting tumor growth and progression in tumor-bearing mice. More importantly, the target receptor expression has been verified in human biopsy samples of breast cancer, indicating that this protein might be a promising new drug target.

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 1625.
Abstract 1628: Tumor cell specific inhibition of oncogenic Wnt signaling
Authors: Takigawa Yutaka , Love Damon K. , Brown Anthony M. C.
Published: February 2011

Aberrant activation of the Wnt/β-catenin pathway is one of the most frequent signaling abnormalities known in human cancer and contributes to both tumor initiation and tumor maintenance. In normal tissues, however, the Wnt/β-catenin pathway plays widespread and important roles in the regulation of stem cell behavior, differentiation, and homeostasis. Consequently, a challenge in developing strategies for antagonizing Wnt signaling in cancer will be to minimize harmful side-effects on normal tissues. In many human cancers the canonical Wnt pathway is activated by single base missense mutations in β-catenin located near N-terminal phosphorylation sites that control the protein's stability. These mutant forms of β-catenin represent targets that are unique to cancer cells. We have screened RNAi reagents designed specifically to target individual mutant β-catenin sequences found in cancer cells while having minimal impact on wild-type β-catenin. We have used colorectal and hepatoma cell lines with defined β-catenin mutations for initial evaluation of the efficacy and specificity of knock-down, in conjunction with signaling assays based on nuclear accumulation of β-catenin and TCF-responsive reporter assays. We find that mutation-specific siRNA or shRNA reagents can be identified that substantially reduce β-catenin levels in cancer cells bearing the respective mutation, but have little or no effect on cells that are wild-type for β-catenin or which carry a different mutation. We also observe mutation-specific inhibitory effects on cell growth. These results provide encouragement for developing this strategy as a means of silencing oncogenic mutations in cancer while causing only minimal collateral damage to normal tissues.

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 1628.
Abstract 1629: Restoration of the wild type function to mutant p53 by chemopreventive agent phenethyl isothiocyanate
Authors: Wang Xiantao , Govind Sudha , Wu Jessie Yu-Chieh , Tomita York , Mi Lixin , Di Pasqua Anthony J. , Chung Fung-Lung
Published: February 2011

p53 mutations occur in more than 50% of all human tumor. Mutant p53 is incapable of activating downstream target genes necessary for cell cycle arrest and apoptosis. Phenethyl isothiocyanate (PEITC), a cruciferous vegetable-derived compound, has been shown to possess anti-cancer property by causing cell growth arrest or apoptosis in tumor cell lines and animal models. Recently, we have found that PEITC can selectively reduce mutant p53, but not wild type protein levels in a variety of tumor cell lines. Here, we demonstrate that PEITC can increase sequence specific DNA binding of p53 in NSCLC H596 cells expressing mutant p53 and induce the expression of p53-dependent target proteins, such as p21 and Noxa. Similar effects were observed in human glioblastoma T98G cells which also express mutant p53. H596 cells incubated with sulforaphane (SFN), another widely studied ITC compound with much weaker cytotoxicity, showed no increased specific DNA binding of p53. In addition, PEITC induced p53-responsive p21 and Noxa promoter luciferase activity. Blocking mutant p53 protein expression with shRNA in T98G cells entirely prevented p21 protein induction triggered by PEITC. Immunoprecipitation studies with conformation-specific p53 antibodies (Pab1620 and Pab240) show that treatment with PEITC alters mutant p53 to wild type conformation in H596 cells. Currently, we are investigating the significance of function restoration to mutant p53 by phenethyl isothiocyanate in the regulation of apoptosis by PEITC or other conventional chemotherapeutic drugs.

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 1629.
Abstract 1633: The sphingosine kinase inhibitor SKI-II blocks survival and MAPK signaling in human breast cancer in vitro
Authors: White Martin D. , Antoon James W. , Meacham William D. , Slaughter Evelyn M. , Burow Matthew E. , Beckman Barbara S.
Published: February 2011

Endocrine resistance is a major contributor to treatment failure in breast cancer. A close examination of the signaling mechanisms by which cells may impart their resistance presents an attractive strategy for the development of viable treatment options. Sphingosine kinase (SK), and its product, sphingosine-1-phosphate (S1P), have been increasingly implicated in many disease states and SK/S1P signaling is a known regulator of apoptosis and cell survival. Furthermore, inhibition of sphingosine kinase has been shown to be anti-proliferative as well as apoptotic in a multitude of human cancer cell lines. In this study, we investigated the biological activity of the sphingosine kinase inhibitor, SKI-II (4-[[4-(4-Chlorophenyl)-2-thiazolyl]amino]phenol) and its ability to disrupt cell signaling in estrogen-dependent and endocrine-resistant breast cancer. SKI-II blocked both viability and survival at low micromolar IC50 concentrations in endocrine therapy-resistant MDA-MB-231, MDA-MB-468 and MDA-MB-361 cells in addition to endocrine therapy-sensitive MCF-7 breast cancer lines. Additionally, treatment with SKI-II significantly (p<0.05) decreased proliferation, as seen in Ki-67 immunostaining. The potent biological activity of this inhibitor led us to characterize the precise molecular mechanisms in which SKI-II imparts its effects. Sphingosine kinase is known to affect various signaling cascades, and its effect on the MAPK and PI3K/Akt pathways are cell line dependent. Growth factor signals such as MAPK can cross-talk with estrogen receptor and confer hormone therapy resistance. Therefore, we chose to investigate several signaling cascades to determine if SK inhibition mediates its effect through disruption of signaling. SKI-II diminished phosphorylation of ERK1/2 in both a time-dependent and dose-dependent manner. Treatment with SKI-II also inhibited phosphorylation of p38/MAPK at similar concentrations. This blockade of MAPK signaling may contribute to SKI-II's dual anti-cancer effect, which can target ER activation in an estrogen-dependent and an estrogen-independent manner. Targeting the production of sphingosine-1-phosphate through pharmacological inhibition presents a potentially useful method to reset the cell signaling aberrations -which lead to endocrine resistance, and may contribute to overall greater efficacy of current therapeutic interventions by re-sensitizing cells to endocrine therapy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1633.
Abstract 1635: Combination of the HDAC inhibitor romidepsin with the MEK inhibitor U0126 results in synergistic plasma cell apoptosis
Authors: Gu Yanyan , Kaufman Jonathan , Boise Lawrence H. , Lonial Sagar
Published: February 2011

Histone deacetylase inhibitors such as vorinostat and panobinostat have been tested as single agents in patients with relapsed myeloma with nominal activity. Yet, when combined with the proteasome inhibitor bortezomib, clinical and laboratory evidence of synergy has been observed. We sought to evaluate the individual signaling effects of the HDACi romidepsin, with the intent of developing additional combinations that may lead to clues of mechanism of action on human myeloma cells.

Single agent studies demonstrated IC50 values for romidepsin alone of between 10 and 30nm, further experiments demonstrate that growth inhibition resulting from romidepsin exposure includes apoptosis and cell cycle arrest. Cell death is associated with increased caspase 3, 8, 9 and PARP cleavage. Cell cycle analysis indicates that romidepsin induces G0/G1 cell cycle arrest which is correlated with p21 upregulation. Cell signaling assessment demonstrates that romidepsin inhibits AKT phosphorylation. Surprisingly, romidepsin increases Erk phosphorylation with its resultant effects on cell growth, development and differentiation. Combining romidepsin with the MEK inhibitor U0126 suppresses MEK/ERK signaling, and synergistically enhances the antitumor effect of romidepsin (combination index value <1). Annexin V staining demonstrates significant apoptosis following 18 hr of treatment with U0126 (5µM) and 10nM romidepsin. Western blot confirms the development of significantly increased procasepase and PARP cleavage, as well as substantial downregulation of Mcl-1, Bcl-xL. The combination can overcome the antiapoptotic effect of exogenous cytokines, such as IL-6 and IGF1 in vitro. Further studies evaluating the effect of bone marrow stromal cells will be presented in addition of data evaluating other potential targets for oncogene addiction that can be used for further novel combinations to gain more understanding of the biological context of this combination both in vitro and in vivo. This study will improve the design of effective clinical combination strategies.

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 1635.
Abstract 1636: Anticancer drug-induced Jak2/Stat3 activation confers a survival advantage in lung cancer cells
Authors: Su Wen-Pin , Lo Ya-Chin , Lin Chien-Chung , Chen Helen H.W. , Lai Wu-Wei , Su Wu-Chou
Published: February 2011

Chemotherapy may trigger survival signaling in cancer cells and the cells may change the response to chemotherapy. Here we found chemotherapeutics-induced Jak2/Stat3 signaling activation, by which cancer cells escape from death in human lung cancer PC14PE6AS2 (AS2) and H157 cells with constitutively activated Stat3 protein. Cisplatin, paclitaxel and epirubicin are important agents in lung cancer treatment and they are reactive oxygen species (ROS)-producing agents. In AS2 and H157 cells, we found that during the paclitaxel treatment, early Jak2 activation occurred and then Stat3 tyrosine phosphorylation upregulated early. But Stat3 serine phosphorylation was not significantly increased. When AS2 and H157 cells were treated with cisplatin and epirubicin, the Jak2/Stat3 activation was also observed, which can be blocked by AG490. It is indicated that chemotherapy, such as paclitaxel, cisplatin and epirubicin, may activate Jak2/Stat3 pathway. In AS2 cells, chemotherapeutics generated ROS, and the chemotherapy-induced Jak2/Stat3 activation was only effectively inhibited by mitochondrial ROS scavengers as rotenone and antimycin. The similar finding was also observed in H157 cells. It is said that “mild” mitochondrial uncoupling provides a survival benefit in cancer cells. In AS2 and H157 cells, pretreatment with the mitochondrial uncoupler carbonylcyanide-4-trifluoromethoxy-phenylhydrazone (FCCP) inhibited paclitaxel-induced mitochondrial ROS generation, abolished paclitaxel-induced Stat3 activation in AS2 and H157 cells, and augmented paclitaxel-induced mitochondrial membrane potential decrease. Thereafter, we classified AS2 and H157 cells as “FCCP-responsive” or “less mitochondrial uncoupling” cancer cells. So that mitochondrial coupling-mediated oxidative stress modulated paclitaxel-induced Jak2/Stat3 activation in AS2 and H157 cells. Subsequently, Stat3 activation by chemotherapeutics enhanced survivin expression in AS2 and H157 cells. Pretreatment with AG490, FCCP and rotenone blocked paclitaxel-induced survivin expression in a dose-dependent manner. Pretreatment with FCCP and rotenone also made AS2 and H157 cells more sensitive to the drug. Conclusively, anti-cancer drugs-induced Jak2/Stat3 activation confers a survival advantage in lung cancer AS2 and H157 cells via mitochondrial-coupling mediated ROS generation. This is a new phenomenon of cellular response to chemotherapeutics, by which cancer cells escape from drug-induced death.

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 1636.