bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4624: Cleavage of HER2 by phenethyl isothiocyanate mediates apoptosis in breast cancer cells
Authors: Gupta Parul , Srivastava Sanjay K.
Published: June 2012

Her2 is a well established marker of breast cancer and found to be overexpressed in around 20-30% of cases. It plays role in cell proliferation, angiogenesis, metastasis and drug resistance leading to poor prognosis and survival. Therefore development of novel agents targeting Her2 is highly imminent. Based on several epidemiological and experimental evidences, isothiocyanates have been shown to have anticancer effects. We evaluated the effect of phenethyl isothiocyanate (PEITC) in breast cancer cells. Treatment with PEITC for 24h significantly suppressed the survival of both MCF-7 (estrogen dependent) and MDA-MB-231 (estrogen independent) cells in a concentration dependent manner with an IC50 of about 12µM and 8µM respectively. Substantial apoptosis by PEITC treatment was observed in both the cell lines. Interestingly, PEITC treatment after 24h drastically reduced Her2 expression. The decrease in Her2 expression by PEITC treatment correlated with the cleavage of Her2. The low molecular weight fragments of Her2 increased with increasing PEITC concentration. Caspase-3 specific inhibitor blocked PEITC-mediated cleavage of Her2 and apoptosis indicating the involvement of caspase-3 in the cleavage of Her2. Her2 fragments have been shown to induce mitochondria-mediated apoptosis. In agreement, our results show the cleavage of Bid and increase in Bax level, whereas the expression of Bcl-XL was down regulated. Further, PEITC treatment resulted in the release of cytochrome c from the mitochondria into the cytosol, activating caspase 9/3 and PARP cleavage. Taken togther, our results suggests that cleavage of Her2 by PEITC activates mitochondrial death pathway in breast cancer cells. Further mechanistic studies are in progress. [Supported in part by R01 grants CA106953 and CA129038 (to S.K.S) awarded by the National Cancer Institute].

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 4624. doi:10.1158/1538-7445.AM2011-4624
Abstract 4646: Occupational exposure to trichloroethylene and plasma concentrations of IL6, IL10, and TNF alpha
Authors: Bassig Bryan A. , Zhang Luoping , Tang Xiaojiang , Vermeulen Roel , Shen Min , Smith Martyn T. , Qiu Chuangyi , Ge Yichen , Ji Zhiying , Reiss Boris , Liu Songwang , Guo Weihong , Purdue Mark , Hu Wei , Li Laiyu , Rothman Nathaniel , Huang Hanlin , Lan Qing
Published: June 2012

Trichloroethylene (TCE) is a chlorinated solvent primarily used as a vapor degreaser in many industries. TCE is currently classified by IARC as probably carcinogenic to humans (Group 2A), and has been associated with non-Hodgkin lymphoma (NHL) in some epidemiological studies, although the findings have been inconsistent. To explore the biologic plausibility that TCE may be a lymphomagen, we conducted a cross-sectional study of workers exposed to TCE in Guangzhou, China and tested if plasma levels of the cytokines IL6, IL10, and TNF-alpha, which play a critical role in regulating various components of the immune system, were altered in exposed workers. We enrolled 80 workers from factories where TCE was used as well as 96 unexposed controls matched by sex and age from food and clothes manufacturing factories. Personal exposure measurements were taken over a three-week period, two to three per subject, in the exposed subjects for a full work shift using a 3M badge, as well as for a subgroup of the control workers. Blood samples were collected from each subject, and plasma concentrations of IL6, IL10, and TNF-alpha were measured by ELISA. Linear regression using the natural logarithm of each endpoint and adjusting for potential confounders was used to test for differences in marker concentrations between controls and exposed workers. The mean TCE exposure level in exposed subjects was 23.9 ppm (95% C.I. 14.2-33.6 ppm). Compared to unexposed workers, the plasma concentration of IL10 in workers exposed to TCE was decreased by more than 50% compared to controls (p=0.003), while there was no difference in levels of IL6 or TNF-alpha in TCE exposed workers and controls. IL10 plays a critical role in mediating the Th1/Th2 balance, apoptosis, and regulation of inflammation. Genotypes associated with altered IL10 expression have also been associated with the development and progression of NHL. Given that immunologic alterations are suspected to play a role in lymphomagenesis, and IL10 plays an important role in immunologic processes, our findings provide some support for the biologic plausibility that TCE is associated with lymphoma.

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 4646. doi:10.1158/1538-7445.AM2011-4646
Abstract 4668: Genetic effects and modifiers of radiotherapy and chemotherapy on survival in pancreatic cancer
Authors: Zeng Hongmei , Yu Herbert , Lu Lingeng , Jain Dhanpat , Kidd Mark S. , Saif M Wasif , Chanock Stephen J. , Hartge Patricia , Risch Harvey A.
Published: June 2012

Purpose: Germline genetic variation may affect clinical outcomes of cancer patients. Using an existing genome-wide association study (GWAS) data set, we applied a candidate-gene approach to evaluate the effect of putative markers on survival of patients with pancreatic cancer. We also examined gene-radiotherapy and gene-chemotherapy interactions, aiming to explain inter-individual differences in treatment outcomes.

Patients, Materials and Methods: In total, 211 patients with pancreatic cancer were identified and recruited between February 2005 and June 2008 in a population-based study and followed through June 2010, an average follow-up of 3.4 years. Sixty-four candidate genes associated with cancer survival or treatment response were selected from existing publications. Genotype information was obtained from a previous GWAS analysis of the study subjects. The main effect of genetic variation and gene-specific treatment interactions on overall survival were examined by proportional hazards regression models.

Results: Fourteen genes showed evidence of association with pancreatic cancer survival. Among these, rs1760217, located at the DPYD gene, rs17091162 at SERPINA3 and rs2231164 at ABCG2 had the lowest P-values of 10−4.60, 0.0013 and 0.0023, respectively. We also observed that two genes, RRM1 and IQGAP2, had significant interactions with radiotherapy in association with survival, and two others, TYMS and MET, showed evidence of interaction with the chemotherapeutic agents 5-FU and erlotinib, respectively.

Conclusions: Our study revealed significant associations between germline genetic polymorphisms and overall survival in pancreatic cancer, as well as survival interactions between various genes and radiotherapy and 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 4668. doi:10.1158/1538-7445.AM2011-4668
Abstract 4683: Inducible knockdown of DNA topoisomerase IIα affects drug sensitivity and cell growth
Authors: Chen Cheng-Fen , He Xiaolong , Beck William T.
Published: June 2012

DNA topoisomerase IIα (Top2α) is an essential nuclear enzyme and target for important anti-cancer drugs, such as etoposide and doxorubicin (Dox). Our teniposide-resistant human leukemic lymphoblastic cells (CEM/VM-1-5) (Danks MK et al., Biochemistry 27(24):8861-8869, 1998) express reduced Top2α protein. To determine the role of Top2α in drug resistance, we knocked down Top2α in parental CEM cells by RNAi. Compared to control cells, CEMshTop2α cells were more resistant to the Top2 inhibitors, etoposide and Dox. The mechanism by which knockdown of Top2α caused resistance to Top2 inhibitors is probably caused by a reduction in Top2-DNA complexes leading to fewer broken DNA strands (Wang JC, Nat Rev Mol Cell Biol 3(6):430-440, 2002). The level of reduced Top2α expression in Top2α knockdown CEM cells was confirmed by Western blot. Importantly, we found no compensatory regulation by Top1 or Top2α overexpression under these conditions. It has been reported that knocking-out of Top2α will cause early embryonic death (Jenkins JR et al. Nucleic acids research 20(21):5587-5592, 1992), but in our cultured CEM cells, knocking Top2α down to 40% of control level had little or no impact on cell growth rate. We subsequently generated and characterized stable CEM-tTR-shTop2α cells where Top2α can be conditionally knocked down to 5% of control levels, as determined by Western blot, through a tetracycline-induced expression of short hairpin RNAs. CEM-tTR-shTop2α cells grow significantly more slowly than the control cells, which indicates that while cells can proliferate with ∼40% of Top2α expression, when Top2α is reduced by 95%, cells can apparently remain viable, but are severely impaired in their ability to grow. Present studies are focused on identifying genes that are targets of Top2α knockdown to provide insights into those genes involved in drug resistance. (Supported in part by CA40570 from NCI and in part by UIC)

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 4683. doi:10.1158/1538-7445.AM2011-4683
Abstract 4711: Silencing of miR-221 with anti-microRNA oligonucleotides is an effective therapeutic for hepatocellular carcinoma
Authors: Park Jong Kook , Kogure Takayuki , Nuovo Gerard J. , He Lei , Kim Ji Hye , Phelps Mitch A. , Croce Carlo M. , Patel Tushar , Schmittgen Thomas D.
Published: June 2012

Patients with advanced hepatocellular carcinoma (HCC) face a dismal prognosis as there are no standardized therapies to treat this stage of the disease. Inhibition of microRNAs that are overexpressed in the tumor with antisense oligonucleotides represents a novel option to treat HCC. For antisense oligonucleotides to be effective, they must reach the target tumor tissue, inhibit the miRNA, modulate the miRNAs target protein levels and prolong survival. We evaluated anti-miR-221 in normal and tumor bearing mice. Of eight different chemistries evaluated, the cholesterol labeled, 2’ O methyl, phosphorothioate modified anti-miR-221 (chol-anti-miR-221) was the most effective at reducing proliferation in vitro. In the absence of transfection reagents, the in vitro proliferation was reduced by the chol-anti-miR-221 but not the scrambled control. Non specific toxicity was observed in vitro for each of the chemically modified scrambled control oligos when transfected with lipid reagent. Chol-anti-miR-221 had improved pharmacokinetics and liver tissue distribution compared to the non cholesterol labeled oligonucleotide in C57Bl/6 mice. Endogenous miR-221 levels in the liver were reduced by chol-anti-miR-221 in C57Bl/6 mice by 90%, 7 days following the start of the dosing schedule. In situ hybridization demonstrated that chol-anti-miR-221 accumulated in the tumor cells of orthotopic mice. Chol-anti-miR-221 reduced endogenous miR-221 levels in the tumors of orthotopic xenograft mice by > 95% and increased p27Kip1 target protein by 3-fold, 7 days after dosing. Chol-anti-miR-221 produced a survival advantage in orthotopic mice compared to those mice treated with a scrambled control oligo (P < 0.05). Our data demonstrate that chol-anti-miR-221 is efficacious in an orthotopic mouse model of HCC and suggests that such a therapy may be beneficial in patients with advanced HCC.

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 4711. doi:10.1158/1538-7445.AM2011-4711
Abstract 4726: Genetic determinants of serum retinol: A genome-wide association study
Authors: Mondul Alison M. , Yu Kai , Weinstein Stephanie J. , Virtamo Jarmo , Jacobs Kevin B. , Kraft Peter , Chanock Stephen J. , Albanes Demetrius
Published: June 2012

Background: Retinol, the most biologically active form of vitamin A, is hypothesized to influence a wide range of diseases. We recently reported results from the Alpha-Tocopherol, Beta-Carotene Cancer Prevention (ATBC) Study suggesting that higher serum retinol is related to increased prostate cancer risk. Circulating retinol concentrations are influenced by many factors in addition to dietary and supplemental intake, including those related to intestinal absorption, transport, and cleavage of pro-vitamin A compounds to form retinol. Thus, genetic factors that influence retinol concentrations may also impact human disease risk.

Methods: We conducted a genome-wide association study (GWAS) of 4,041 Caucasian individuals drawn from two cohorts: the ATBC Study and the Prostate, Lung, Colorectal, and Ovarian (PLCO) Cancer Screening Trial. Our findings were replicated in an independent sample of 2,056 individuals from the Nurses’ Health Study.

Results: We identified two independent SNPs near the genes encoding key retinol carrier proteins, transthyretin (TTR) and retinol binding protein 4 (RBP4), that were associated at the level of genome-wide significance with retinol concentrations: rs1667255 (in 18q12.1) and rs10882272 (in 10q23.33). After meta-analysis with the replication sample, these SNPs remained highly significant (rs1667255: p = 2.33 × 10-14; rs10882272: p = 1.08 × 10-13). In the two GWAS studies, mean serum retinol concentrations increased with number of risk alleles (0,1,2) for TTR from 556-578-602 μg/L in ATBC and 654-688-711 μg/L in PLCO, and decreased for 0,1,2 risk alleles in RBP4 (580-567-541 μg/L in ATBC and 697-668-653 μg/L in PLCO).

Conclusions: This is the first GWAS of circulating retinol levels to reveal novel, genome-wide significant loci, one near TTR and one near RBP4, genes that encode the two major retinol carrier proteins. Understanding the genetic and other determinants of retinol status may help shed light on the etiology of cancer and other diseases associated with vitamin A.

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 4726. doi:10.1158/1538-7445.AM2011-4726
Abstract 4738: Cellular resistance screening with a novel selective c-Met inhibitor reveals a spectrum of missense mutations that partially overlap with activating mutations found in cancer patients
Authors: Tiedt Ralph , Degenkolbe Elisa , Furet Pascal , Appleton Brent A. , Schoepfer Joseph , Ruddy David , Monahan John , Jones Michael D. , Blank Jutta , Haasen Dorothea , Drueckes Peter , Wartmann Markus , McCarthy Clive , Hofmann Francesco
Published: June 2012

Emergence of resistance is a major concern when treating cancer patients with targeted kinase inhibitors, as exemplified by the appearance of point mutations in Bcr-Abl in CML patients treated with imatinib or a secondary EGFR-T790M mutation in lung cancer patients treated with gefitinib or erlotinib. In vitro approaches to identify resistance mutations in Bcr-Abl have yielded mutation spectra that faithfully recapitulated clinical observations.

In order to predict resistance mutations in the receptor tyrosine kinase c-Met that could emerge during inhibitor treatment in patients, we performed a resistance screen in BaF3 cells transformed with Tpr-Met using the novel selective c-Met inhibitor NVP-BVU972. The observed spectrum of mutations in resistant cells was dominated by substitutions of the residue Y1230 in the activation loop of the c-Met kinase domain, but also included other missense mutations. Intriguingly, some of the c-Met resistance mutations observed in this cellular screen were identical with previously described activating mutations in cancer patients that interfere with inhibitory interactions in the inactive conformation. Co-crystallization of the c-Met kinase domain in complex with NVP-BVU972 revealed a key role for Y1230 in binding of NVP-BVU972. This binding mode has also been reported for multiple other selective c-Met inhibitors, some of which have entered clinical trials, suggesting a broader relevance of the resistance profile obtained with NVP-BVU972.

A second resistance screen in the same format with the c-Met inhibitor AMG 458 yielded a distinct spectrum of mutations that was rich in F1200 alterations. This is consistent with a different predicted binding mode. Again, the mutation profile observed with AMG 458 could be predictive for several other inhibitors that bind c-Met in a similar way.

Collectively, our findings suggest that amino acid substitutions in the c-Met kinase domain of cancer patients need to be carefully monitored prior to and during treatment with c-Met inhibitors, as resistance may pre-exist or emerge. Compounds binding in the same manner as NVP-BVU972 might be particularly susceptible to the development of resistance through mutations in Y1230, a complication that could potentially be overcome by c-Met inhibitors with alternative binding modes.

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 4738. doi:10.1158/1538-7445.AM2011-4738
Abstract 4752: Cortactin and coronin 1B cooperate to promote tumor cell invasion in head and neck squamous cell carcinoma
Authors: Walk Elyse L. , Wu Hong , Garcia Marileila V. , Bear James E. , Weed Scott A.
Published: June 2012

Amplification of chromosome 11q13 in head and neck squamous cell carcinoma (HNSCC) is a common occurrence associated with poor patient prognosis. Within the core amplified region is the gene that encodes cortactin (CTTN), a filamentous (F)- actin binding protein and Src kinase substrate that stabilizes Arp2/3-F-actin branchpoints in dynamic cortical F-actin structures. CTTN amplification results in increased lymph node metastases and decreased survival rates in HNSCC patients. CTTN amplification in HNSCC cell lines enhances tumor cell motility and invasion. Chromosomal regions of varying length flanking the core 11q13 amplicon are often amplified in HNSCC, and contain several genes with the potential to cooperate with CTTN in regulating actin dynamics. One of these genes within the 11q13 region outside of the core amplicon is CORO1B (coronin 1B), an actin-binding protein that antagonizes cortactin function by promoting the breakdown of cortactin-stabilized Arp2/3-F-actin branches, resulting in impaired cell motility. Here we show that coronin 1B is a potential mediator of HNSCC invasion. CORO1B amplification and overexpression occurs in a subset of HNSCC patients with CTTN amplification. Coronin 1B localizes with cortactin within HNSCC invadopodia, ventral membranous protrusions responsible for degrading extracellular matrix to facilitate loco-regional invasion and extracapsular spread. RNAi-mediated coronin 1B knockdown decreases the number of invadopodia per cell, enhances cell spreading and reduces extracellular matrix degradation. These results collectively point to a potential role for coronin 1B in regulating HNSCC tumor cell invasion through modulating cortactin activity and invadopodia function. Our data also support a potential diagnostic function for CORO1B amplification in denoting a previously unidentified HNSCC subset that may display increased invasiveness in cases containing 11q13 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 4752. doi:10.1158/1538-7445.AM2011-4752
Abstract 4753: The interaction of two metastasis suppressor genes, Nm23-H1 and Gelsolin, results in synergistic phenotypes
Authors: Marino Natascia , Marshall Jean-Claude A. , Collins Joshua , Zhou Ming , Veenstra Timothy , Steeg Patricia S.
Published: June 2012

Metastasis Suppressor Genes (MSGs) are able to inhibit metastasis without reducing primary tumor size. Nm23 was the first MSGs to be identified. The biochemical basis for the anti-metastatic effect of Nm23 has been subject of many studies but remains unresolved. We performed co-immunoprecipitation assays using flag-tagged constructs of both the human and murine forms of Nm23 (Nm23-H1 and Nm23-M1, respectively) over-expressed in the mouse mammary carcinoma cell line 4T1. Co-immunoprecipitation assays were also performed on tissue extracts of primary tumors and metastases obtained from Balb/C mice injected with these cell lines. Using Mass-Spectrometry, new binding partners were identified including the actin-binding protein Gelsolin. Gelsolin is an actin regulatory protein that modulates actin assembly and disassembly, and is believed to regulate cell motility in vivo through modulation of the actin network. Recently, Gelsolin has been added to the list of MSGs.

An interaction between Gelsolin and Nm23 was validated in a murine cell line (4T1- Nm23-H1/M1 clones) and in a human breast carcinoma cell lines (MCF-7). In the low- metastatic cells MCF-7 an interaction between endogenous Nm23-H1 and Gelsolin was observed. However, in the highly metastatic MDA-MB-435 and MDA-MB-231 cell lines the interaction was observed only after the over-expression of Nm23-H1. These results suggest that an Nm23-Gelsolin interaction may only take place when relatively high levels of Nm23 are expressed. We conclude that the interaction between Nm23-H1 and Gelsolin occurs in the non-metastatic state, where high levels of both proteins are present, and this interaction can contribute to the anti-metastatic property associated to Nm23.

Using cultures of MDA-MB-231 stably overexpressing Gelsolin, Nm23-H1 or both proteins, the biological role of this interaction was analyzed. The two metastasis suppressors, Nm23-H1 and Gelsolin, when overexpressed in the single stable clone did not affect the cell growth; however they cooperated in decreasing the cell growth. This is the first time overexpression of Nm23-H1 has decreased cell proliferatioin and suggests that combinations of MSGs may have distinct phenotypic effects. Moreover, overexpression of both Nm23-H1 and Gelsolin in MDA-MB-231 cells reduced cell migration and cell invasion compared to the clone overexpressing the empty vector or single suppressor genes. In summary, Nm23-H1 has been demonstrated to bind another Metastasis Suppressor, Gelsolin. Additive and synergistic effects of multiple Metastasis Suppressors were identified.

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 4753. doi:10.1158/1538-7445.AM2011-4753
Abstract 477: CD49fhi/CD90lo from primary human osteosarcoma cells are enriched for tumor initiating property
Authors: Penfornis Patrice , Cai David Z. , Harris Michael R. , Induru Spandana , Meyer Mark , Pochampally Radhika R.
Published: June 2012

Osteosarcoma (OS) is one of the most prolific forms of cancer among adolescents, resulting in a severe decrease in life expectancy unless detected early. Though several previous studies have suggested various tumor-initiating properties of osteosarcoma, major limitations for understanding have been observed due the OS's restrictive ability to expand in in vivo models. In this study, we are reporting the use of primary osteosarcoma cells for characterizing the tumor initiating property. Human OS samples were collected during biopsies or surgeries, expanded in vitro, phenotyped for multiple cell-surface markers by Fluorescence Activated Cell Sorting (FACS) analysis and then injected subcutaneously into nu/nu mice. We observed that cells showing an expression of the CD49fhi/CD90lo cell surface marker produced a more aggressive tumor. In vitro treatment of this cell population with amiloride, a potassium-sparing diuretic, decreased the cells ability to express the CD49f protein. In vivo, amiloride treatment also dramatically decreased tumor growth when injected into nu/nu mice when compared to controls, indicating that CD49fhi/CD90lo is an important marker in tumor initiation. The transfection of CD49fhi cells with a shCD49f lentiviral construct showed a 20% decrease in their overall ability to migrate in a trans-well assay, and experienced a two-fold decrease in their capability to form colonies (CFU assay). Subcutaneous injections of the transfected cells expressing shRNA for CD49f reduced tumor growth by 30% compared to controls. In conclusion, our data suggests that the CD49fhi/CD90lo cell population is linked to a more aggressive osteosarcoma tumor growth and that this cell surface marker could be a potential candidate for the characterization of TICs in osteosarcoma.

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 477. doi:10.1158/1538-7445.AM2011-477
Abstract 4775: Role of FoxM1 survival pathway in primary effusion lymphoma cells
Authors: Ahmed Maqbool , Hussain Azhar R. , Ahmed Saeeda O. , Bu Rong , Thangavel Saravanan , Platanias Leonidas C. , Al-Kuraya Khawla S. , Uddin Shahab
Published: June 2012

Primary effusion lymphoma (PEL) is associated with Kaposi sarcoma herpesvirus (KSHV) but its pathogenesis is poorly understood. Many KSHV-associated products can deregulate cellular pathways commonly targeted in cancer. The clinical features and natural history of PELs differ greatly from those observed in other lymphomas. The failure to improve outcomes with treatment intensification indicates the need for the introduction of new therapeutic options. Forkhead Box M1 (FoxM1) is a transcription factor that regulates the expression of a number of genes that are involved in cell cycle regulation and metastasis. Over-expression of FoxM1 has been shown in a variety of cancer however its role has not been fully elucidated in PELs. We found that FoxM1 is constitutively expressed in PELs and treatment of PELs with thiostrepton; a specific inhibitor of FoxM1 inhibits cell viability and induces apoptosis in a dose dependent manner. In addition, thiostrepton treatment also efficiently blocks formation of colonies on agarose plates as well as inhibits cell invasion and migration of PEL cell. Thiostrepton treatment causes down-regulation of FoxM1, MMP-2 and MMP-9 in PEL cells leading to activation of the intrinsic apoptotic pathway. Thiostrepton treatment causes conformational changes of Bax protein, leading to loss of mitochondrial membrane potential and release of cytochrome c to the cytosole. This leads to activation of caspase-9, caspase-3, and polyadenosin-5’-diphosphate-ribose polymerase (PARP) cleavage leading to caspase-dependent apoptosis. Altogether, this data demonstrates the possible role of FoxM1 in pathogenesis of PELs and raise the possibility that incorporation of thiostrepton in treatment regimens for primary effusion lymphomas may improve the overall survival of patients suffering from this malignancy.

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 4775. doi:10.1158/1538-7445.AM2011-4775
Abstract 4801: Comprehensive genome methylation analysis in bladder cancer; identification and validation of novel methylated genes and application of these as urinary tumor markers
Authors: Reinert Thomas , Modin Charlotte , Lamy Philippe , Wojdacz Tomasz K. , Hansen Lise L. , Wiuf Carsten , Borre Michael , Dyrskjøt Lars , Ørntoft Torben F.
Published: June 2012

Cancer of the urinary bladder is one of the major cancers being the fifth most common neoplasm in the industrialised countries. Urothelial carcinomas account for more than 90% of all bladder cancers. The majority of bladder cancers are non-muscle invasive cancers characterized by frequent recurrences after resection and up to 25% will develop an aggressive phenotype. We investigated the methylation in bladder cancer with respect to location in genome, consistency, variation in metachronous tumors, impact on transcripts, chromosomal location, and usefulness as urinary markers of bladder cancer.

In this study, a microarray assay with more than 27.000 CpG sites was utilized to analyze methylation in 56 samples. Independent validations were performed in 63 samples by methylation-sensitive high-resolution melting (MS-HRM) and bisulfite sequencing. Transcript levels were analyzed using expression microarrays, and pathways using dedicated software. DNA from 40 urinary specimens was analyzed by MS-HRM.

Our results show that global methylation patterns were established within and outside CpG-islands. Differential methylation (p<0.0001 to p<0.05) was identified in 1588 genes between normals and cancers, in 1019 genes with stage specific methylation, and in 149 genes as progression markers. We validated methylation of eight genes, the tumor markers ZNF154 (p<0.0001), HOXA9 (p<0.0001), and POU4F2 (p<0.0001) and the progression marker TBX4 (p<0.002). The methylation of metachronous tumors was relatively stable and targeted to certain pathways. The correlation to expression was not stringent. Chromosome 21 showed most differential methylation (p<0.0001) and specifically hypomethylation of keratins, which together with keratin like proteins were epigenetically regulated. In DNA from voided urine we detected differential methylation of ZNF154 (p<0.0001), POU4F2 (p<0.0001), and HOXA9 (p<0.0044), achieving 94% sensitivity and 100% specificity.

Our results suggest that both hypo- and hyper methylation is stage dependent, over-represented at chromosome 21, and has a restricted influence on transcript levels in general, but clearly on keratins. The methylated genes are highly promising urinary cancer markers.

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 4801. doi:10.1158/1538-7445.AM2011-4801
Abstract 4806: Claudin downregulation in head and neck squamous cell carcinoma (HNSCC) may be caused by aberrant promoter methylation
Authors: Nagado Aline A. , de Carvalho Ana Carolina , Lessa Roberta C. , Lourenço Silvia V. , Coutinho-Camillo Claudia M. , Buim Marcilei E.C. , Soares Fernando A. , Carvalho André L. , de Oliveira André Luiz Vettore
Published: June 2012

Claudins are a family of transmembrane proteins that compose the tight junctions, playing an essential role in cell-to-cell adhesion, maintenance of cell polarity and recruiting signaling proteins. The expression of these proteins has been found to be altered in several human cancers, suggesting that this family of proteins might represent potential molecular markers for cancer detection, diagnosis and therapy. We analyzed the expression profile of a claudin member gene in Head and Neck Squamous Cell Carcinomas (HNSCC) cases using Real-Time PCR, and found low expression in 78% of the evaluated cases. The expression of this gene was restored in an HNSCC cell line after treatment with the demethylating agent 5-Aza-2′-Deoxycytidine. After that, we designed primers for methylation analysis (MSP) and examined the expression and methylation status of this gene in several cell lines. A positive correlation between the expression level and methylation pattern was found. Besides, preliminary analyses of aberrant methylation status in HNSCC and normal samples showed 100% of specificity and 24% of sensitivity. This result suggests that aberrant methylation of claudin members could play a major role in the expression control of this gene.

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 4806. doi:10.1158/1538-7445.AM2011-4806
Abstract 4817: Altered DNA methylation in human lung cell line A549 after treatment with a standard mixture of particulate matter
Authors: Rota Federica , Dioni Laura , Cantone Laura , Baccarelli Andrea , Bertazzi Pier Alberto
Published: June 2012

BACKGROUND: Numerous health studies have shown acute and chronic particulate air pollution (PM, particulate matter) exposures to be associated with lung cancer, as well as with lung growth impairment, chronic obstructive pulmonary disease (COPD), and asthma. Toxicology studies have shown that PM is only a weak mutagen. Whether PM induces DNA methylation alterations, a primary mechanism in cell transformation, has never been studied in lung epithelial cells. In our study, we found a significative modification of P16 DNA methylation after PM treatement.

AIM: To investigate PM on 8 cancer and inflammatory genes (TLR-4, CD14, IFNγ, TNFα, RASSF1A, APC, p53, p16) in A549 human alveolar basal epithelial cells.

METHODS: A549 cells were treated with a complex standard mixture of particulate matter (NIST-National Institute of Standards and Technology- Standard Reference Material [SRM] 1648a). SRM 1648a treatments were performed with increasing doses of total mixture (15, 31, 62, 125, and 250 μg/ml) dissolved in colture medium. We used untreated cells as a negative control. Only concentrations that did not affect cell viability were considered. The cells were harvested after 2, 6, 12, 24, and 48 hours of continuous exposure, washed with phosphate buffered saline, and stored as cell pellet at -80°C until DNA extraction. DNA methylation was measured by means of bisulfite-Pyrosequencing. All the experiments were conducted in triplicate to confirm repeatability of our results.

RESULTS: Using a t-test assuming that the two distributions have the same variance, we found an increase in p16 methylation for a dose of 15 μg/ml (p16 mean(untreated-treated)=0,83; p-value=0,05); an hypomethylation for a dose of 62 μg/ml (p16 mean(untreated-treated)=−0,77; p-value=0,004) as well as for a dose of 250 μg/ml (p16 mean(untreated-treated)=−0,78; p-value=0,01). We observed also an increase in APC methylation for a dose of 250 μg/ml (APC mean(untreated-treated)=2,90; p-value=0,02). CONCLUSION: This finding indicates that different levels of exposure to PM can induce changes in the DNA methylation levels in vitro. Our hypothesis is that exposure to PM can cause changes in the gene-specific DNA methylation levels thus enhancing cancerogenesis. Additional studies are required to better define the epigenetic mechanisms by which PM and similar environmental agents induce changes in DNA methylation.

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 4817. doi:10.1158/1538-7445.AM2011-4817
Abstract 482: High CD44/CD24 expressive cells presented cancer stem cell characteristics and undergo mitochondrial resetting and metabolic shift in nasopharyngeal carcinoma
Authors: Shen Yao-An , Wei Yau-Huei , Chen Yann-Jang
Published: June 2012

Cancer stem cells (CSCs) are a rare subset cancer cells with self-renewal property and cause many difficulties in clinical treatment. CSCs have been found in different tumors by specific surface markers. However, the surface marker of CSC remains unknown in various cancer types, including in nasopharyngeal carcinoma (NPC). Here, we took advantage of CSC's properties, such as chemoresistance, radioresistance, and the ability to form tumor spheres, to isolate CSCs from NPC cell lines. Combining these three isolation methods, and surface marker screening, we identified that NPC CSCs had high level of CD44 and CD24 surface antigen expression. We then validated the CD44/CD24 high positive as NPC CSC surface marker. In cell proliferation assay by MTT test, tumorigenity assay in soft agar and in vivo xenotransplantation, and radioresistant assay, CD44/CD24 high positive cells expressed significant CSC properties compared with CD44/CD24 double negative and parental NPC cells. We further found that CSCs underwent mitochondria resetting, including the distribution, quantity, and quality of mitochondria. Metabolic shift that largely relied on glycolysis also occured in CSCs. They predominately ultilized glycolysis instead of mitochondrial respiration to meet energy demand. Inhibiting glycolysis or treating antioxidants suppresses the viability and malignant traits of CSCs which provides therapeutic implication in NPC.

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 482. doi:10.1158/1538-7445.AM2011-482
Abstract 4821: Somatic variation scoring and validation for large-scale cancer genome sequencing
Authors: Pant Krishna P. , Lincoln Steve , Baccash Jonathan , Halpern Aaron L. , Nazarenko Igor , Carnevali Paolo , Ballinger Dennis G.
Published: June 2012

Complete Genomics has sequenced the genomes of over 100 tumor-normal sample pairs from a variety of cancers (e.g. Lee, et al., Nature 465: 473-477, 2010) using a unique high throughput sequencing platform (Drmanac, et al., Science 327:78-81, 2010). We have assembled a tool set to investigate somatic mutations in these samples, including small variations (SNVs, deletions, insertions and substitutions), as well as larger structural variations, copy number variations and regions of LOH (Loss of Heterozygosity). Included in this sample set are 12 cancer cell lines and matched normal cell lines obtained from the American Type Tissue Culture collection (ATCC), comprising 10 breast cancers and 2 lung cancers. We have developed and characterized somatic variation scoring methods for each variant class that can be tuned for particular applications and sample types. Comparative analyses with previously published data show high specificity and sensitivity of somatic variation detection. We will discuss the application of these methods for somatic variation analysis in larger cancer genome studies.

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 4821. doi:10.1158/1538-7445.AM2011-4821
Abstract 4848: Genetic profile of invasive breast cancer vasculature using laser capture microdissection (LCM) to capture vessels
Authors: Jones Dylan T. , Lechertier Tanguy , Herbert John , Bicknell Roy , Jones Louise J. , Harris Adrian L. , Hodivala-Dilke Kairbaan
Published: June 2012

Tumor angiogenesis is vital for tumor growth and progression. Defining genes that are involved in tumor driven angiogenesis may yield therapeutically important targets for cancer therapy.

We have developed and optimized a rapid 25 minute immunohistochemical staining method using CD31 antibody (Pecam1) specific to endothelial cells in vessels on sections from frozen human breast cancer (n=3) and normal breast (n=3), followed by laser capture microdisection (LCM) of vessels. Good quality RNA was extracted amplified, and fluorescently labeled cDNA was synthesized and hybridized onto Affymetrix U-133 Plus 2.0 array.

Using this methodology, we were able to elucidate the genetic profile of tumor associated vessels from invasive breast cancer and compare it with vessels in normal breast tissue. This technique has the advantage over other methods in analyzing genetic profile of tissue as it analyses the genetic profile of isolated cell population and eliminates genes expressed by surrounding tissue, allowing for the detection of low expression of genes that were not detected in whole tumor arrays.

Using false discovery rate statistical analysis (q<0.05) of microarray data, we identified 64 upregulated and 6 downregulated genes in breast cancer associated vessels. With Ingenuity Pathway analysis the top molecular and cellular functions include 11 genes involved in Cellular Assembly and Organization, 9 in Cellular Function and Maintenance and 8 in Protein Synthesis. Canonical pathway analysis revealed 3 genes involved in Ephrin Receptor signaling, 3 in Integrin signaling, 2 in VEGF signaling and 2 in TGF-β signaling. Differentiated genes were compared to Expression Sequence Tags from endothelial and non-endothelial libraries; three genes were specific to endothelial cells only (q<0.05). Using Immunohistochemistry we have validated expression of two genes in breast vessels.

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 4848. doi:10.1158/1538-7445.AM2011-4848
Abstract 4890: Diffusion-weighted MRI provides an early assessment of treatment response in a murine model of metastatic prostate cancer to the bone
Authors: Brisset Jean-Christophe , Galbán Stefanie , Rehemtulla Alnawaz , Pienta Kenneth J. , Galbán Craig J. , Ross Brian Dale
Published: June 2012

Ninety percent of patients with advanced prostate cancer develop metastases to the bone1. At present, there is no definitive means for assessing response to therapy, with RECIST identifying tumors in the bone as “unmeasureable”. Diffusion-weighted MRI (DW-MRI) is highly sensitive to microscopic changes in tumor tissue. Our goal is to use DW-MRI to detect and quantify changes in tumor tissue throughout therapy where the magnitude of the change could be related to the effectiveness of the treatment. Human prostate cancer (PC3) cells were implanted by direct intra-tibial injection into male SCID mice, which served as a model for prostate bone metastases. MRI experiments were performed on a 9.4T MRI. Upon identifying a tumor volume of ∼10mm3, animals were randomly distributed into 4 treatment groups: docetaxel 20 mg/kg i.p. dose once a week for 3 doses (qd7×3; N=5), fractionated radiation IR at 2 Gy/day 5x per week for 2 weeks (N=7), combined treatment (N=5) and control (N=6). Animals were imaged by DW-MRI in which the apparent diffusion coefficient was calculated from the DW images. Tumor volume and ADC measurements were acquired pre-treatment and on days 0, 1, 4, 7, and once per week after post-treatment initiation. Animals were removed from the study when the tumor reached a percent increase of 400% (5x the initial tumor volume). Group comparisons in MRI metrics were obtained using an unpaired Student t-test. Mice treated by Dox or IR had significant smaller increase in tumor volume compared to control mice. Combined treatment resulted in improved efficacy over lone therapies. This resulted in a significant decrease in tumor size. The Dox group showed an increase in ADC following the first administration and a slow decline in ADC values following day 4 post-treatment initiations regardless of additional treatments. ADC value for IR treated animals did not elevate above control until day 7. Combined treatment generated increased ADC values that were maintained throughout the treatment cycle. IR and Dox alone and in combination were effective as assessed by anatomical and DW-MRI. Those results demonstrate the potential of quantitative ADC map for assessing early response of metastatic cancer to the bone.

References:

1. Carlin BI et al. Cancer. 2000;88(12):2989-2994. 2. Lee KC et al. Cancer Res. 2007;67(8):3524-3528. 3. Lee KC Neoplasia. 2007;9(12):1003-1011.

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 4890. doi:10.1158/1538-7445.AM2011-4890
Abstract 4895: Tagging endogenous genes with fluorescent reporters using CompoZr® zinc finger nuclease technology
Authors: Zhang Hongyi , Zhang Fan , Zenser Nathan , Vassar Deborah L. , Malkov Dmitry , Davis Gary
Published: June 2012

Zinc finger nucleases (ZFNs) are a class of engineered DNA-binding proteins that facilitate targeted editing of the genome by creating double-strand breaks (DSBs) in DNA at desired locations. DSBs are important for site-specific mutagenesis in that they stimulate the cell's natural DNA-repair processes, namely homologous recombination (HR) and non-homologous end joining (NHEJ). The formation of a DSB increases the rate of HR by a thousand fold between a specific genomic target and a donor plasmid in somatic cells. Sigma-Aldrich CompoZr® ZFN technology is a useful tool to create site specific mutations, and to generate knock-in and knockout model lines for academic and clinical research.

By this approach, we tagged an oncogene, several cytoskeletal genes and a chromatin gene by integrating a fluorescent protein sequence into the desired location (at the sequences encoding either N-terminus or C-terminus of the target protein) in the genome. The integration resulted in endogenous expression of the corresponding fusion proteins (green, red, or blue) that shows their native characteristic pattern. The following five loci were tagged: ERBB2/HER2 (human epidermal growth factor receptor 2, plasma membrane), TUBA1B (α-tubulin 1b, microtubule), ACTB (β-actin, actin stress fibers), LMNB1 (lamin B1, nuclear envelope) and HMGA1 (high mobility group AT-hook 1, nucleus). Single cell clones were isolated in SKOV3, U2OS, and MCF10 cells with one copy of a given gene tagged. Multiplexing was demonstrated by labeling different genes in the same cell line as well as different alleles of the same gene.

We demonstrate that ZFN mediated gene tagging is an efficient way to generate knock-in cell lines to report endogenous gene expression. It provides the basis for development of various high content screening (HCS) assays for compound screening where target gene regulation and corresponding protein function are preserved.

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 4895. doi:10.1158/1538-7445.AM2011-4895