bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 822: Withaferin A inhibits breast cancer invasion and metastasis at subcytotoxic doses by inducing vimentin disassembly and serine 56 phosphorylation
Authors: Thaiparambil Jose T. , Bender Laura , Ganesh Thota , Kline Erik , Patel Pritty , Liu Yuan , Tighiouart Mourad , Vertino Paula M. , Harvey R Donald , Garcia Anapatricia , Marcus Adam
Published: June 2012

Withaferin A (WFA) is purified from the plant Withania somnifera and inhibits the vimentin cytoskeleton. Vimentin overexpression in cancer correlates with metastatic disease, induction of epithelial to mesenchymal transition, and reduced patient survival. Since WFA has significant anti-tumor activity and vimentin functions in cell motility, we wanted to test the hypothesis that WFA inhibits cancer metastasis by disrupting vimentin function. These data show that WFA has weak antiproliferative and apoptotic activity at concentrations less than or equal to 500 nM, but retains potent anti-invasive activity at these low doses. Live cell imaging in breast cancer cell lines revealed that WFA induces perinuclear vimentin bundling followed by rapid vimentin depolymerization. A concomitant induction of vimentin ser56 phosphorylation was observed, which is consistent with vimentin disassembly. Structure activity relationships were established using a set of chemically modified WFA analogs and showed that the predicted vimentin-binding region of WFA is necessary to induce vimentin ser56 phosphorylation and for its anti-invasive activity. Pharmacokinetic studies in mice revealed that WFA reaches peak concentrations up to 2μM in plasma with a half-life of 1.36 hours following a single 4 mg/kg dose. In a breast cancer metastasis mouse model, WFA showed dose-dependent inhibition of metastatic lung nodules and also induced vimentin ser56 phosphorylation, with minimal toxicity to lung tissue. Based upon these studies, we conclude that WFA is a potent breast cancer anti-metastatic agent and the anti-metastatic activity of WFA is, at least in part, mediated through its effects on vimentin and vimentin ser56 phosphorylation.

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 822. doi:10.1158/1538-7445.AM2011-822
Abstract 828: Design, synthesis and biological evaluation of peptide inhibitors of the Keap1-Nrf2 protein-protein interaction
Authors: Hancock Rowena , Bertrand Helene , Naz Shama , Tsujita Tadayuki , Hayes John , Wells Geoff
Published: June 2012

Background: Compounds that block the cellular effects of carcinogens have the potential to be cancer chemopreventive agents. A number of dietary constituents such as sulforaphane found in broccoli and curcumin found in turmeric increase the expression of antioxidant response element (ARE) genes involved in detoxifying reactive primary metabolites. The mode of action of these chemopreventive agents has been determined as an interaction with cysteine residues in the protein Keap1, this interaction causes Keap1 to release its binding partner, the transcription factor Nrf2. The free Nrf2 then translocates to the nucleus and up-regulates ARE dependent genes resulting in cellular protection against reactive species (McMahon et al., 2003).

Aims: Our aim is to develop compounds that directly inhibit the Keap1-Nrf2 protein-protein interaction and thereby enhance Nrf2 activity by a mechanism different to that of existing chemopreventive agents. Such compounds will have the potential to be useful molecular probes for studying the Keap1-Nrf2 interaction and the Nrf2 signaling pathway in detail and also as candidate preventive agents for cancer.

Methods: A series of linear peptides based on the high affinity binding motif of Nrf2 (the ETGE sequence) were designed, synthesized and screened to determine the optimum chain length for β-turn formation and interaction with the Keap1 protein. A second generation of peptides with changes to the sequence were synthesized to improve the binding profile. The peptides were synthesized using standard Fmoc chemistry and were purified by preparative HPLC, then lyophilized prior to use. The peptides were characterized by 1H-NMR, HPLC, LC-MS and high resolution MS.

Results: The peptides were synthesized successfully and were shown to cause inhibition of the Keap1-Nrf2 protein-protein interaction, determined using a fluorescence polarization assay developed in our laboratory. A number of the sequence change peptides have equivalent or superior activity to the native Nrf2 sequence.

Conclusion: In vitro tests have shown that the peptide sequence, structure and length are important for inhibition of the protein-protein interaction and the data have allowed us to propose an initial structure activity relationship for the Keap1-peptide interaction. The results and their implications for future drug design will be presented.

Reference: McMahon, M., Itoh, K., Yamamoto, M., Hayes, J. (2003) J. Biol. Chem. 278, 21592-21600

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 828. doi:10.1158/1538-7445.AM2011-828
Abstract 836: Rexinoids induce cellular senescence in ER-negative breast cancer cells and in MMTV-Neu mammary tumors
Authors: Shilkaitis Anne , Green Albert , Bratescu Laura , Peng Xinjian , Steele Vernon , Christov Konstantin T.
Published: June 2012

Retinoids and recently rexinoids have been extensively studied as potential cancer preventive agents. Their effects both in vitro and in vivo have been associated with inhibition of cell proliferation via cell cycle regulatory genes/proteins. In previous studies we have shown that retinoids (atRA, 9cRA), in addition to inhibition of cell proliferation, can also induce cellular senescence (CS) in breast cancer cell lines and in an MNU-model of mammary carcinogenesis in rats. We also found that atRA is more efficacious in inducing CS in ER+ than in ER− breast cancer cell lines.

Here we examined the role of rexinoids (LGD1069) in inducing CS in ER− breast cancer cell lines and in ER− tumors of the MMTV-Neu model of mammary carcinogenesis in mice. CS was determined by SA-β-Gal staining, colony formation assay, heterochromatin condensation and histone H2AX expression. Cells grown on cover slips were treated for 6 days with 1.0 or 5.0 µM of LGD1069 and senescent cells identified. We found that LGD1069 at 1.0 µM is more efficacious in inducing CS in ER+ MCF-7 and T47D cells, 56% and 42% respectively vs. 14% and 9% in control cells. Normal MCF10A, premalignant MCF10AT, and malignant MDA-MB-231 and MDA-MB-468 cells, which are ER−, required 5.0 µM of LGD1069 to increase CS from 3.0, 6,0, 4.0 and 10.0% in control to 14, 24, 26 and 33% in treated cells. For in vivo studies, MMTV-Neu mice with palpable mammary tumors were treated for 4 weeks with 80 or 40 mg/kg body weight LGD1069, given by gavage. Proliferating cells (BrdU-labeled) in mammary tumors decreased from 9.6±2.3% in control animals (No=13) to 7.4±3.0% (p<0.01) (No=10) and to 5.6±2.5% (p<0.001) (No=12) in high and low dose treated animals, respectively. Senescent cells, as detected by SA-β-Gal staining, were rare in control tumors (0.92±0.23%) and significantly increased in LGD1069, 80 mg/kg treated animals (6.8±4.0%) (p<0.001). LGD1069 was unable to affect apoptosis. Our mechanistic studies suggest involvement of p21, RXRα and RARβ in mediating LGD1069-induced CS in ER− breast cancer cells.

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 836. doi:10.1158/1538-7445.AM2011-836
Abstract 844: Acyclic retinoid inhibits diethylnitrosamine-induced liver tumorigenesis in obese and diabetic C57BL/KsJ-db/db mice
Authors: Shimizu Masahito , Sakai Hiroyasu , Shirakami Yohei , Yasuda Yoichi , Kubota Masaya , Moriwaki Hisataka
Published: June 2012

Background and aims: Obesity and the related metabolic abnormalities are associated with increased risk of hepatocellular carcinoma (HCC). Malfunctioning of retinoid X receptor (RXR)-α due to phosphorylation by Ras/MAPK also plays a critical role in liver carcinogenesis. In the present study, we examined the effects of acyclic retinoid (ACR), which targets RXRα, on the development of diethylnitrosamine (DEN)-induced liver tumorigenesis in C57BL/KsJ-db/db (db/db) obese and diabetic mice. Methods: Male db/db mice were given tap water containing 40 ppm DEN for 2 weeks, after which they were fed a diet containing 0.03% or 0.06% of ACR throughout the experiment. Results: In mice treated with either dose of ACR for 34 weeks, the development of liver cell adenomas was significantly inhibited as compared to basal diet-fed mice. ACR markedly inhibited the activation of Ras and phosphorylation of the ERK, RXRα, and Stat3 proteins in the livers of experimental mice. It also increased the expression of RARβ and p21CIP1 mRNA, while decreasing the expression of cyclin D1, c-Fos, and c-Jun mRNA in the liver, thereby restoring RXRα function. Administration of ACR improved liver steatosis and activated the AMPK protein. The serum levels of insulin were decreased by ACR treatment, whereas the QUICKI values were increased, indicating improved insulin sensitivity. The serum levels of TNF-α and the expression levels of TNF-α, IL-6, and IL-1β mRNA in the livers of DEN-treated db/db mice were decreased by ACR treatment, suggesting attenuation of the chronic inflammation induced by excessive fatty deposits. Conclusion: ACR may be, therefore, useful in the chemoprevention of obesity-related 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 844. doi:10.1158/1538-7445.AM2011-844
Abstract 85: Detection of promoter methylation of tumor supressor genes in serum DNA of breast cancer patients and controls
Authors: Sturgeon Susan R. , Balasubramanian Raji , Schairer Catherine , Ziegler Regina , Muss Hyman , Arcaro Kathleen
Published: June 2012

Background: Changes in DNA methylation patterns are a common feature of malignant cells. Methylation is an epigenetic change that affects gene expression. Methylation of a cytosine guanine dinucleotide-rich area (CpG island) of the promoter region of a gene that is normally unmethylated is correlated with silencing of that gene. Certain tumor suppressor genes are hypermethylated in breast tumor tissue and rarely methylated in normal breast tissue. Circulating serum DNA, presumably including DNA shed from the tumor, can be tested for promoter hypermethylation. Breast cancer detection with moderate sensitivity has been achieved in several small studies relying on a few genes and suggests that assaying methylation levels in a larger panel of genes might result in a useful serum marker for breast cancer detection.

Methods: Bisulfite-modified serum DNA was evaluated for promoter methylation via pyrosequencing in 12 candidate tumor suppressor genes (GSTP1, SRFP1, RAR-beta, ER1, TWIST, HIN1, APC, ECAD, p16, BRAC1, CyclinD2, RASSF1). Study subjects included approximately 250 node-positive postmenopausal breast cancer cases, 75 node negative postmenopausal breast cancer cases and a comparison group of 250 postmenopausal women with benign breast disease types not strongly associated with breast cancer, all of whom donated blood at the time of diagnosis to the Mayo Serum Bank, a resource established in the 1970's to identify early markers of breast cancer.

Results: As hypothesized, mean percent levels of serum DNA methylation were modestly but statistically higher in node-positive breast cancer cases than controls for TWIST (4.9% vs 3.9%; p-value =.002), CyclinD2 (3.2% vs 2.1%; p=.03), and HIN1 (3.9% vs 2.4%; p=.03). In the nine other genes, percent methylation levels were also moderately higher in node-positive cases than controls but did not reach statistical significance (e.g., SRFP1 [6.3% vs 4.3%; p=.06]). For TWIST, Cyclin D2, HIN1, mean percent levels were intermediate in node-negative patients compared to node-positive cases and controls. Additional analyses evaluating the predictive ability of multivariate sets of DNA methylation markers together with established breast cancer risk factors are underway.

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 85. doi:10.1158/1538-7445.AM2011-85
Abstract 854: Alternative dosing regimens with the EGFr inhibitors (Gefitinib and Lapatinib) in a mammary cancer model: Prevention efficacy
Authors: Lubet Ronald A. , Bode Ann , Szabo Eva , Grubbs Clinton J.
Published: June 2012

The EGFR inhibitors (Gefitinib, Lapatinib) have proven to be effective in preventing cancers in multiple animal models, including ER and Neu positive breast, head and neck, and urinary bladder cancers when administered daily. However, the development of toxicities (Iressa, skin rashes; Lapatinib, diarrhea) limit their potential use in prevention; and perhaps even in an adjuvant setting. We, therefore, tested whether alternative dosing regimens (which might reduce toxicity) would still achieve preventive and therapeutic efficacy. Female Sprague-Dawley rats were administered a single IV dose of methylnitrosourea (MNU) at 50 days of age. In the prevention study, rats were administered Gefitinib by gavage beginning five days after MNU. MNU treated rats were administered Gefitinib daily (10 mg/kg BW/day, 7x/week) or Gefitinib (70 mg/kg BW, 1x/week). These treatment regimens resulted in 94 and 75% reductions in tumor multiplicity, respectively. Simultaneous measurements of tumor load (number of tumors x tumor weight) showed that both regimens resulted in greater than a 90% decrease. In the therapeutic study (initiating treatment when animals developed a small palpable tumor), both regimens were highly effective. A similar prevention study was performed with Lapatinib (75 mg/kg BW/day, 7x/week or 525 mg/kg BW, 1x/week). While the daily dose reduced cancer multiplicity 90%, the weekly dose caused a 70% reduction. These data show that even a great alteration of the dosing of Gefitinib (EGFR1) or Lapatinib (EGFR 2/1) resulted in a large reduction in mammary cancers. The significant clinical question will be whether this altered dosing will diminish the toxicities associated with these agents. We cannot determine this in the animal model since, at the effective doses employed, the typical toxicities observed in humans were not observed. Supported by NCI contract number HHSN261200433001C.

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 854. doi:10.1158/1538-7445.AM2011-854
Abstract 866: D,L-Sulforaphane-induced apoptosis in human breast cancer cells is regulated by growth factor adaptor protein p66Shc
Authors: Sakao Kozue , Singh Shivendra V.
Published: June 2012

D,L-Sulforaphane (SFN), a synthetic racemic analogue of broccoli constituent L-sulforaphane, inhibits chemically-induced cancer in experimental rodents. SFN administration also suppresses cancer development in transgenic mouse models. Anticancer effect of SFN is associated with apoptosis induction but the mechanism of cell death is not fully understood. Using MDA-MB-231 (an estrogen-independent cell line) and MCF-7 (an estrogen-responsive cell line) human breast cancer cells as a model, we now demonstrate that the SFN-induced apoptosis is regulated by growth factor adapter protein p66Shc. Exposure of MDA-MB-231 and MCF-7 cells to SFN resulted in increased Ser36 phosphorylation of p66Shc. Immortalized mouse embryonic fibroblasts derived from p66Shc knockout mouse were significantly more resistant to SFN-mediated growth inhibition (judged by trypan blue dye exclusion assay) and apoptosis induction (judged by cytoplasmic histone-associated apoptotic DNA fragmentation) compared with embryonic fibroblasts from wild-type mouse. Moreover, transient transfection of MDA-MB-231 and MCF-7 cells with p66Shc-targeted siRNA conferred significant protection against SFN-induced cytoplasmic histone-associated DNA fragmentation. Protein level of prolyl isomerase 1 (Pin1), a protein implicated in mitochondrial translocation of p66Shc, was decreased on a 24-hour treatment with SFN in both MDA-MB-231 and MCF-7 cells. However, apoptosis induction resulting from SFN exposure was not affected by ectopic expression of Pin1 (MCF-7). In conclusion, the present study provides novel insights into the molecular circuitry of SFN-induced apoptosis involving p66Shc. This investigation was supported by the USPHS grant RO1 CA115498-05, 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 866. doi:10.1158/1538-7445.AM2011-866
Abstract 871: A stratified genetic risk assessment for testicular cancer
Authors: Kratz Christian P. , Greene Mark H. , Shi Jianxin
Published: June 2012

Three recent genomewide association studies (GWAS) of testicular germ cell tumors (TGCT) have uncovered predisposition alleles in or near several genes including KITLG, BAK1, SPRY4, TERT, ATF7IP and DMRT1. A candidate study has identified the so-called AZFc region as another risk locus on chromosome Y. We used a statistical approach to dissect the question of whether TGCT risk alleles can be utilized in the clinical setting. We employed the receiver operating characteristic curves for genetic risk models to measure the discriminatory power of a genetic risk model. The newly-discovered variants provided a discriminatory power of 69.2%, which was considerably higher than for other cancers analyzed with this method. This suggested that about 69.2% of the time, a randomly-selected patient with TGCT had a higher estimated risk than the risk for a randomly-selected control subject. Using a multiplicative model, we estimated that white men in the top 1% of genetic risk had a relative risk that was 10.5-fold greater than that for the general white male population in the US. Under the assumption that clinical risk factors such as cryptorchidism are not correlated with TGCT risk variants and do not interact with the TGCT loci as contributors to TGCT risk, white men aged 30-34 years with a history of cryptorchidism who were in the top 1% of genetic risk had an estimated 5-year risk of 4.4%. Other clinical risk factors such as family history and infertility could be also used in a stratified genetic risk assessment approach. Extensive statistical modeling and additional genotyping is required before such accurate risk estimates are feasible. We conclude that a genetic risk assessment is not justified in the general population at this time. However, a stratified genetic risk assessment strategy may be useful for men who have independent clinical risk factors.

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 871. doi:10.1158/1538-7445.AM2011-871
Abstract 88: Promoter hypermethylation leads to loss of Wnt7a in NSCLC
Authors: Tennis Meredith A. , VanScoyk Michelle , Freeman Scott , Winn Robert
Published: June 2012

The Wnt signaling pathway is critical to normal development but the involvement of the pathway in lung tumorigenesis has not been clearly established. Wnt7a, with its receptor Fzd9, is required for maintenance of epithelial differentiation and inhibits transformed growth. Wnt7a has been shown to be frequently lost in NSCLC cell lines and tumors. We demonstrate that loss of Wnt7a in a non-transformed lung epithelial cell line leads to increased proliferation. Using pyrosequencing, 58 of 81 (72%) of NSCLC tumors we tested had higher methylation in the Wnt7a promoter region compared to adjacent normal lung tissue. We also tested 10 pairs of human NSCLC tumor and normal adjacent lung samples with known loss of expression of Wnt7a in the tumor and 6 (60%) had increased methylation of the Wnt7a promoter region in the tumors. Two NSCLC cell lines demonstrated to have methylated Wnt7a and loss of Wnt7a expression, H157 and H1299, were used to investigate methylation of the Wnt7a promoter. Treatment of these cell lines with 5-aza-2deoxycytidine resulted in increased expression of Wnt7a and increased downstream activity of known targets of Wnt7a-Fzd9 signaling. Methylation of a Wnt7a promoter luciferase resulted in decreased activity of the luciferase compared to an untreated Wnt7a luciferase. NNK is a tobacco carcinogen known to attenuate DNMT1 degradation, leading to increased promoter methylation. Treatment of a non-transformed lung epithelial cell line with NNK resulted in decreased expression of Wnt7a and increased clonogenicity. Together, these results indicate that Wnt7a is frequently lost by methylation in NSCLC and that methylation of Wnt7a leads to decreased expression. Wnt7a could be methylated by exposure to tobacco carcinogens, leading to loss of expression and increased transformed lung cell growth. The clinical use of demethylating agents could restore Wnt7a activity, helping to decrease growth of NSCLC.

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 88. doi:10.1158/1538-7445.AM2011-88
Abstract 882: Copy number variation analysis identified multiple genetic variants related to nasopharyngeal carcinoma predisposition
Authors: Su Wen-Hui , Tse Ka-Po , Yang Min-lee , Shugart Yin Yao , Chang Yu-Sun
Published: June 2012

Nasopharyngeal carcinoma (NPC) is a multi-factorial malignancy closely associated with genetic factors and Epstein-Barr virus infection. Recently, we have conducted a genome-wide association study (GWAS) in 277 NPC patients and 285 healthy controls within the Taiwanese population, analyzing 480,365 single-nucleotide polymorphisms (SNPs). Twelve statistically significant SNPs were identified and mapped to chromosome 6p21.3 in the major histocompatibility complex region. In addition to HLA-A gene, we detected novel significant associations between NPC and two genes: specifically, gamma aminobutyric acid b receptor 1 and HLA-F. To our knowledge, it is the first GWAS report of NPC showing that multiple loci (HLA-A, HLA-F, and GABBR1) within chromosome 6p21.3 are associated with NPC. Following the GWAS study, we made an effort to search systematically for copy number variations (CNVs) that confer increased risk to NPC using the same dataset. We generated CNV calls using four different softwares and used 15 internal duplicated samples to look for best filtering criteria. CNVs were selected if same CNVs were identified from two independent softwares to increase credibility of prediction. We identified eight CNVs that overrepresented in NPC patients compared to healthy controls. Six deletions on chromosomes 3, 6, 7, 8 and 19, as well as 2 duplications on chromosomes 7 and 12 were found. One of the most significant CNV on chromosome 6p21.3 was further replicated in an independent case-control dataset, using a deletion-specific polymerase-chain reaction. To the best of our knowledge, it is the first study of genome-wide constitutional CNV 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 882. doi:10.1158/1538-7445.AM2011-882
Abstract 896: A systematic search in tumor interstitial fluid identifying a tumor-specific interleukin-hypoxia-glycolysis-DNA repair pathway and a novel serum marker RBB3 for human hepatoma
Authors: Hsieh Sen-Yung
Published: June 2012

The long-term outcome of patients with hepatocellular carcinoma (HCC) remains undesirable, primarily due to late diagnosis and high recurrence. We aimed to discover new markers for early detection and prediction of recurrence. We initiated our search by comparing the proteomes of the interstitial fluid of HCC (TIF) and nonHCC liver tissues by using proteomic approaches. About 150 proteins were found to be dysregulated in TIF. Gene ontology analysis and biofunction reconstruction revealed the involvement of “interleukin-hypoxia-glycolysis-DNA repair pathway” in HCC. To search for novel serum markers, we identified secreted RBB3 (sRBB3) significantly increased their amount in TIF. Further studies disclosed that the serum sRBB3 level was significantly higher in HCC (113 cases) than in cirrhosis (47 cases)(p < 0.001) and chronic hepatitis (64 cases)(p < 0.001). The accuracy of serum sRBB3 in detection of HCC was further validated in 2 independent sets (92 cases in each set) of patients by using receiver operating characteristic (ROC) curve. A sensitivity of 89.3% and a specificity of 99.7% were obtained. Moreover, serum srbb3 had a better performance than serum alpha-fetoprotein (AFP) in discrimination of early HCC from chronic hepatitis [areas under the ROC curves (AUC) for srbb3 vs. AFP = 0.931 vs. 0.810] and from cirrhosis (AUCs: serbb3 vs. AFP = 0.709 vs. 0.627). The serum sRBB3 level was strongly associated with portal vein invasion and extrahepatic metastasis of HCC (p = 0.017). The performance of the combination of serum srbb3 and AFP was similar to that of srbb3 but better than that of AFP in discrimination of HCC from non-HCC. Conclusion: Interstitial fluid of tumor tissues is a good source in the search for novel tumor markers. An immune-mediated tumor microenvironment plays roles in genome damage and possibly tumorigenesis of HCC. Serum srbb3 is a better HCC marker than AFP, particularly for detection of early HCC and for portal vein invasion and extrahepatic metastatsis.

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 896. doi:10.1158/1538-7445.AM2011-896
Abstract 899: TMPRSS2:ERG urine test identifies Gleason score upgrading and significant prostate cancer
Authors: Aubin Sheila M. , Tomlins Scott , Meyer Sarah , Penabella Yvonne , Siddiqui Javed , Wei John , Chinnaiyan Arul , Rittenhouse Harry , Groskopf Jack
Published: June 2012

Objective: In men diagnosed with prostate cancer (PCa) at biopsy, a major challenge is correctly stratifying those with indolent versus significant PCa for treatment selection. The extent and grade of PCa is assessed in part based on standard risk factors, biopsy Gleason score (bx GS) and the number of positive biopsy cores. However, due to under-sampling at biopsy, upgrading in Gleason score is often seen from biopsy to prostatectomy. In this study we evaluated a TMPRSS2:ERG (T2:ERG) urine test for its ability to identify significant cancer and upgrading at prostatectomy.

Methods: Post-DRE urine specimens were prospectively collected from 218 men referred for prostatectomy. T2:ERG mRNA copies were quantified using a transcription-mediated amplification assay and normalized to PSA mRNA copies to calculate a T2:ERG score. The prototype T2:ERG urine assay detects the gene fusion mRNA isoform TMPRSS2 exon 1 to ERG exon 4. T2:ERG score was correlated to prostatectomy Gleason score (px GS) and significant cancer as defined by the Epstein criteria at prostatectomy.

Results: Of 185 men scheduled for radical prostatectomy, 61 had low risk of significant cancer based on having bx GS<6 and % positive biopsy cores<33%, yet upon prostatectomy, 57% of these men were found to harbor a significant PCa. The T2:ERG assay identified an additional 31% prostatectomy significant cancer in the biopsy low-risk disease cancer group. In a multivariate model, adding T2:ERG to bx GS and % positive cores significantly increased the accuracy for predicting significant PCa at prostatectomy from 0.84 to 0.89 (p=0.0002). Sensitivity increased by 8% for detecting prostatectomy significant cancer when T2:ERG was added to these biopsy criteria (sens/spec=85%/87% vs 77%/87%). T2:ERG also significantly correlated with Gleason score upgrading from biopsy to prostatectomy (bx GS/px GS = 6/ 6 vs bx GS/px GS = 6/>7, p = 0.0076). At biopsy, 70 men were found to have low-grade cancer (bx GS<6), but upon prostatectomy, upgrading was seen in 50% of these men. T2:ERG was able to identify 40% of men with bx GS<6 that were later upgraded.

Conclusions: A T2:ERG urine test may help identify significant cancers and Gleason score upgrading, and increase predictive accuracy when used in combination with currently available methods.

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 899. doi:10.1158/1538-7445.AM2011-899
Abstract 9: 2-deoxyglucose caused toxicity is regulated by the Bcl-2 family and is enhanced by Bcl-2 antagonist in lymphoma cell lines
Authors: Zagorodna Oksana , Martin Sean , Kuwana Tomomi , Spitz Douglas , Knudson C. Michael
Published: June 2012

Introduction. 2-deoxyglucose (2DG) is a glucose analog and inhibitor of glycolysis that has been shown to have anti-cancer activity, proposed to be due to altered metabolism of cancer cells. 2DG has also been reported to induce ER stress through inhibition of N-linked glycosylation. Whether 2DG is effective in lymphoma and the mechanisms of cell death are not known.

Materials and Methods. Five tumor cell lines were independently derived from Lck-Bax driven murine T cell lymphomas (TCLs). The sensitivity of these cell lines to 2DG was determined by plasma membrane integrity and clonogenic survival. Caspase inhibitors, overexpression of Bcl-2, downregulation of Bim, the Bcl-2 antagonist ABT-737, and mannose were utilized to determine the molecular mechanism of 2DG-caused death in lymphoma cell lines.

Results and Conclusions. TCLs are uniformly sensitive to physiologically relevant concentrations of 2DG treatment. The cell death pathway involves Bax “activation” and Caspase cleavage following 2DG treatment. However, qVD-OPh effectively inhibited caspase activity while providing minimal protection from death. In contrast, expression of Bcl-2 enhanced both short term and clonogenic survival of 2DG treated cells. A Bcl-2 antagonist, ABT-737, sensitized TCLs to 2DG-induced cell death. Examination of BH3-only members demonstrated that both Bim and Bmf were upregulated following 2DG treatment. Importantly, downregulation of Bim with shRNAs protected from 2DG treatment, demonstrating that Bim is a critical mediator of 2DG toxicity. We also observed that 2DG led to CHOP induction, a transcription factor shown to directly mediate Bim upregulation following ER stress. Mannose, a reagent known to alleviate ER stress induced by 2DG, also transiently protected from cell death. While mannose effectively blocked CHOP upregulation, Bim upregulation was delayed but not blocked indicating that 2DG upregulated BIM and induced cell death by ER stress dependent and independent mechanism. However, both pathways converge at a step that involves the activation of Bax and is blocked by Bcl-2. We conclude that 2DG toxicity is regulated by the Bcl-2 family, and the combination of Bcl-2 inhibition and 2DG is a candidate for effective therapeutic strategy for 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 9. doi:10.1158/1538-7445.AM2011-9
Abstract 921: HER3 reactivation: A compensatory shift between Akt-mediated positive and negative feedback loops in HER2-overexpressed human breast cancer cells
Authors: Tang Patrick Ming Kuen , Leung Fenix , Gijsen Merel , Harris Adrian L. , Kong Anthony
Published: June 2012

The activated serine/threonine protein kinase Akt (or Protein Kinase B, PKB) regulates cell survival and metabolism by phosphorylation of numerous adaptor proteins and kinases. Akt is known as a key downstream target of HER3 signaling axis. It is also suggested to be an important modulator for the HER3 reactivation via a compensatory shift in the HER3 phosphorylation-dephosphorylation equilibrium during TKI-induced drug resistance. However, the mechanism of Akt-mediated HER3 reactivation during TKI treatment is still unclear. Interestingly, HER3 was rapidly dephosphorylated when an Akt inhibitor (Akt inhibitor VIII, Akti) was applied to mimic the inhibitory effect of TKI on the PI3K/Akt axis. The same phenomenon was also found when PI3K inhibitor was utilized instead. This positive feedback within the HER3 signaling axis was shown to be mediated by PTPN9, a phosphatase that is known to negatively regulate the EGFR activation state. We showed that PTPN9 was upregulated once Akt was inhibited. This lead to EGFR deactivation and eventually caused the dephosphorylation of HER3, which was reversed when PTPN9 was locked down. Furthermore, reactivation of HER3 was also found in the Akti-treated cells at 24h. This was shown to be due to a negative feedback loop via the ADAM17-mediated heregulin release, which could be suppressed by ADAM17 inhibitor INCB4298. Our results are crucial in advancing our knowledge in HER3/PI3K/Akt axis, where the positive loop revealed the mechanism of Akt suppression in regulating HER3 phosphorylation; and the negative loop is implicated in resistance mechanisms to various targeted therapies that affect Akt activity.

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 921. doi:10.1158/1538-7445.AM2011-921
Abstract 928: Species and cell type independent core MYC target genes in cancer and stem cells
Authors: Ji Hongkai , Wu George , Zhan Xiangcan , Nolan Alexandra , Koh Cheryl , De Marzo Angelo , Fan Jinshui , Cheadle Chris , Fallahi Mohammad , Cleveland John , Dang Chi V. , Zeller Karen
Published: June 2012

The c-Myc (hereafter Myc) transcription factor augments the induction of pluripotency of somatic cells and contributes to human tumorigenesis, but the mechanisms by which Myc mediates these phenotypes remain only partially understood. To decipher Myc's biological functions, it is essential to define its target genes. To date, numerous Myc target gene lists have been constructed in various cellular contexts from several species but no study has rigorously defined species and cell type independent core Myc targets. Using a model of human B lymphoma cells bearing inducible MYC, we identified direct Myc target genes via chromatin immunoprecipitation (ChIP), global nuclear run-on assay, and changes in mRNA levels. We also identified, for the first time, direct Myc targets in human embryonic stem cells (ESCs). Here we report that Myc regulates a core set of target genes that are shared in mouse and human ESCs as well as in four other human cancer cell types. The expression of this gene set is increased in Myc-expressing premalignant B lymphocytes and is highly elevated in lymphoma cells from Eμ-Myc transgenic mice. Remarkably, the expression of the core gene set significantly correlates with MYC expression across 8,129 microarray samples in Gene Expression Omnibus (GEO), which represent 312 cell and tissue types. Expression profiles of B cell lymphomas, acute leukemia, lung cancers and Ewing sarcomas are among those with the highest correlation. Annotation of this gene signature highlights Myc's primordial function in ribosomal biogenesis as one of its key roles in cancer and stem cells.

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 928. doi:10.1158/1538-7445.AM2011-928
Abstract 94: Wnt signaling antagonists are progressively hypermethylated during colorectal neoplastic progression
Authors: Silva Ana-Luisa , Arends Mark J. , Wyllie Andrew H. , Ibrahim Ashraf EK
Published: June 2012

Background: Colorectal cancer (CRC) is the third most common type of cancer. The Wnt signaling pathway is known to play an important role in initiation and progression of CRC. Suppression of Wnt antagonists by promoter hypermethylation has been proposed to increase Wnt activity. DNA methylation changes of Wnt antagonists during CRC progression through the preinvasive adenoma stage have not been previously characterized. We analyzed promoter methylation changes of selected Wnt antagonists in a set of synchronous normal, hyperplastic or adenomatous polyps and adenocarcinoma tissue samples obtained from colectomy specimens.

Method: We profiled methylation changes in CpG islands associated with the Wnt signaling antagonists DKK1, DKK2, DKK3, WIF1, SFRP1, SFRP2, SFRP4, SFRP5, and SOX17 using pyrosequencing assays. We also examined methylation changes of two members of the β-catenin destruction complex, APC and AXIN2, and two non-antagonist genes (CDH1 and DVL2) involved in Wnt signaling. The sample set included 48 cases comprising matched normal (n=73), hyperplastic polyp (n=9), adenoma (n=42) and adenocarcinoma (n=48) tissue samples.

Results: Our samples showed significant widespread hypermethylation changes of a subset of the analyzed Wnt antagonists in the transition from normal to hyperplastic polyp to carcinoma and from normal to adenoma to carcinoma, mostly in an increasing stepwise pattern (p<0.05, Wilcoxon rank sum test). DKK1 showed no significant change during CRC progression, DKK3 did not show significant methylation changes during the hyperplastic polyp to carcinoma stage and SFRP4 was not significantly hypermethylated during adenoma to adenocarcinoma progression. APC was hypermethylated in a small subset of carcinomas, however, these changes were not statistically significant. AXIN2 and the non-antagonist Wnt genes, CDH1 and DVL2 showed consistent normal levels of methylation throughout CRC progression.

Conclusion: Wnt antagonists are progressively hypermethylation during CRC neoplastic development. We propose that CpG island hypermethylation of Wnt antagonists could be used as biomarkers for early detection of CRC.

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 94. doi:10.1158/1538-7445.AM2011-94
Abstract 960: Ceramide-dependent release of nanovesicles is a class effect oflipid-based Akt inhibitors
Authors: Gills Joell J. , Zhang Chunyu , Abu-Asab Mones S. , Castillo S. Sianna , Marceau Caleb , Kozikowski Alan , Tsokos Maria , Goldkorn Tzipora , Dennis Phillip A.
Published: June 2012

Akt regulates many cellular processes and Akt inhibitors are being developed as cancer therapeutics. The mechanism of action of lipid-based Akt inhibitors (LBAIs) such as perifosine and phosphatidylinositol ether lipid analogues (PIAs) is incompletely understood. Here, we show that LBAIs quickly deplete cells of proteins such as EGFR, IGFR and p-Akt that can be recovered in tetraspanin-enriched 100,000 xg pellets from conditioned media. Electron micrographs of these pellets exhibited heterogenous vesicular structures whose diameter was less than 1 µM. In vitro, LBAI treatment increased vesicle shedding as revealed by live cell microscopy. In vivo, LBAIs increased plasma levels of nanovesicles derived from tumor xenografts. Several lines of evidence show that release of nanovesicles by LBAI depends upon ceramide generation. LBAIs increased levels of cellular ceramide, and addition of exogenous ceramide stimulated nanovesicle release. GW4869, an inhibitor of sphingomyelinase, an enzyme that generates ceramide by cleavage of sphingomyelin, blocked release of nanovesicles induced by LBAI. When transferred in vitro, purified nanovesicles increased ceramide levels and inhibited proliferation of naïve recipient cells, suggesting these contain residual LBAIs. Pre-treatment with GW4869 blocked ceramide induction in recipient cells and yielded greater retention of the donor nanovesicles. These studies identify ceramide-dependent release of cellular nanovesicles as a class effect of LBAI and a novel means to shed EGFR, IGF-IR and p-Akt from tumors that could perpetuate the cytotoxicity of LBAIs. We believe this is the first report of secondary pharmacologic effects via cell-mediated drug transfer in nanovesicles.

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 960. doi:10.1158/1538-7445.AM2011-960
Abstract 97: Chronic nicotine and NNK exposure causes promoter hypermethylation of GAD2 in human pancreatic duct epithelial cells
Authors: Bernert Heike , Plummer Howard K. , Schuller Hildegard M.
Published: June 2012

Epigenetic alterations are present at early stages of carcinogenesis, and DNA promoter hypermethylation in precursor lesions potentially affects transcriptional regulation. Previous studies by our laboratory have shown that the inhibitory neurotransmitter γ-aminobutyric acid (GABA) inhibits the proliferation of human pancreatic cancer cells grown both in vitro and in mouse xenografts. To test the hypothesis that nicotine as well as the nicotine-derived nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) induce changes in methylation status of the GABA synthesizing enzymes GAD1 (GAD67) and GAD2 (GAD65), the human pancreatic duct epithelial cell line clone HPDE6-E6E7c7 has been exposed to daily treatment of either nicotine or NNK over a course of five days. This initial screen of the methylation status of the promoter region of GAD1 and GAD2 by bisulfate genomic sequencing in HPDE6-E6E7c7 indicates aberrant methylation of GAD2 by either nicotine or NNK. Quantitative methylation-specific PCR as well as gene expression studies by real-time PCR after 5-aza-2’-deoxycytidine treatment, alone or in combination with trichostatin A on the last day of treatment along with concurrent five day treatment with nicotine or NNK confirm these observations that aberrant methylation of GAD2 may be an important factor in the pathogenesis of pancreatic cancer. Quantitative methylation-specific PCR indicates aberrant methylation in six out of ten human matched normal versus cancerous pancreatic tissue samples, which corroborates the in vitro results. In conclusion, exposure to nicotine and NNK leads to significant epigenetic alterations of the GAD2 promoter region. Consecutive silencing of GAD2 with suppression of GABA synthesis may represent an important mechanism by which nicotine and related compounds induce disturbances in inhibitory neurotransmitter circuits which may contribute to the pathogenesis of ductal adenocarcinoma of the pancreas. Supported by grants 1RO1CA130888 and 2RO1CA042829 with 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 97. doi:10.1158/1538-7445.AM2011-97
Abstract 976: Hypoxic microenvironment promotes breast cancer stem cells through regulation of Polycomb protein expression
Authors: Chang Chun-Ju , Hung Mien-Chie
Published: June 2012

The microenvironment of solid tumors contains regions of poor oxygenation as a result of hypoxia, which is associated with high grade breast cancer and poor prognosis. However, critical regulatory mechanism linking tumor microenvironment to cancer progression remains unclear. Cancer stem cells (tumor initiating cells) are suggested to be responsible for initiation and aggressive progression of cancer. It is recently proposed that an aggressive secondary cancer stem cell population arises from a primary cancer stem cell population through acquisition of additional genomic/genetic mutations and drives cancer progression. Additionally, high expression of a Polycomb protein EZH2, essential in stem cell self-renewal, has been linked to aggressive progression of breast cancer. Here, we discovered that hypoxic microenvironment upregulates HIF1α-mediated transcriptional activation of EZH2 in CD44+CD24−/low breast tumor initiating cells (BTICs) isolated from several human primary tumor samples. Enhanced EZH2 expression was required for the hypoxia-promoted BTICs, where hypoxia significantly increased BTIC abundance, tumor sphere formation and tumor growth in mammary fat pads implanted with less than 500 primary BTICs. Using cDNA expression array and genome wide SNP array analysis, we further identified a novel mechanism in which enhanced expression of EZH2 by hypoxia downregulates DNA damage repair proteins through H3K27 methylation and thereby allows the acquisition of recurrent genomic abnormalities such as RAF1 gene amplification in BTICs. Using proteomic analysis, we demonstrated that RAF1 amplification highly activates its downstream ERK-β-catenin signaling, which leads to enhanced survival and proliferation of the BTICs. Furthermore, using immunohistochemistry analysis in a cohort of breast tumor samples, we found that EZH2 expression is positively correlated with RAF1, p-ERK and β-catenin expression in the high grade, poorly-differentiated tumors but not in the low grade, well-differentiated tumors, suggesting EZH2-amplified RAF1-ERK-β-catenin signaling may contribute significantly to promote BTICs and aggravate breast cancer malignancy. Our data also reveal that AZD6244, a clinical trial drug that inhibits RAF1-ERK signaling, can significantly suppress EZH2-promoted BTICs. To the best of our knowledge, this is the first study that provides evidences linking a specific genomic aberration mediated by EZH2-impaired DNA damage response to expansion of BTICs. Together, this study elucidates a previously unidentified therapeutic effect of RAF1-ERK signaling inhibitors to prevent breast cancer progression by eliminating BTICs.

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 976. doi:10.1158/1538-7445.AM2011-976