bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4834: Microarray gene expression analysis on formalin-fixed, paraffin embedded material is feasible and the resulting profiles are comparable to profiles from fresh frozen matched material
Authors: Mittempergher Lorenza , de Ronde Jorma J. , Nieuwland Marja , Kerkhoven Ron M. , Simon Iris , Wessels Lodewyk FA , Van ‘t Veer Laura J.
Published: June 2012

Formalin Fixed Paraffin Embedded (FFPE) samples represent a valuable resource for cancer research and clinical setting. However, the discovery and development of new cancer biomarkers often requires fresh frozen (FF) samples. In this study, we profiled, in duplicate, 20 FFPE breast cancer tissues and 20 matched FF tissues and evaluated the concordance of the gene expression results from FFPE and matched FF material using the whole Genome (WG) DASL assay. We show that clinically relevant biomarkers developed on FF samples can be applied to FFPE samples. We show that after proper normalization, all FFPE and FF pairs exhibited a high level of similarity (Pearson correlation > 0.7), significantly larger than the similarity between non-paired samples. Interestingly, the probes showing the highest correlation had a higher G/C content percentage and were enriched for cell cycle genes. Predictions of gene expression signatures developed on frozen material (Intrinsic subtype, Genomic Grade Index, 70 gene prognosis signature) show a high level of concordance between FFPE and FF matched pairs. Predictions based on a 60 gene DASL list (best match with the 70 gene signature) show very high concordance with MammaPrint® results. We demonstrate that data generated from FFPE material with the DASL assay, if properly processed, are comparable to data extracted from the FF counterpart. This opens up the possibility of using both FFPE and FF material in gene expressions analyses, leading to a vast increase in the potential resources available for cancer research.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4834. doi:10.1158/1538-7445.AM2011-4834
Abstract 485: Hypoxia increases the expression of stem-cell markers and promotes cell invasion in prostate cancer cells
Authors: Bae Kyungmi , Dai Yao , Vieweg Johannes , Siemann Dietmar
Published: June 2012

Oxygen is an essential regulator of cellular metabolism, survival, and proliferation. Cellular responses to oxygen levels are monitored, in part, by the transcriptional activity of the hypoxia inducible factors (HIFs). The existence of hypoxic microenvironment is a common occurrence in many solid tumors including prostate. Previous studies from our laboratories have shown that in human prostate cancer models, cancer stem cells expressing the embryonic stem cell markers SOX2 and OCT3/4 were highly invasive and, importantly, were capable of altering their E-cadherin expression during the process of invasion. In contrast, non-tumorigenic cells which minimally express SOX2 or OCT3/4 were found to be poorly invasive. Furthermore, targeted knockdown of SOX2 or OCT3/4 markedly suppressed the invasion of prostate cancer cells, suggesting an indispensible role of these two stem cell markers in invasiveness. In the present study, we examined the impact of oxygen deprivation on cell invasiveness and stemness in both stem cell enriched and non-stem cell populations in PC3 and DU145 prostate cancer cells. Moreover, the impact of Notch signaling, which has been shown as a critical regulator of prostate cancer development, progression and metastasis, was examined. When cells were exposed to 1% oxygen for 24 h, the invasive capacity of both the stem- and non-stem cell subpopulations increased. Interestingly the non-stem cell subpopulation showed the most striking increase (> 10-fold) in invasion under hypoxic conditions. The expression of several important stem cell factors, including OCT3/4, SOX2, Nanog and c-Myc, also was upregulated under hypoxia in both subpopulations. In addition, the expression of Notch1, Notch2 and their target genes Hey1, Hey2 and HeyL were increased in both subpopulations that had experienced oxygen deprivation (1% O2, 24 h). These findings suggest that although cancer stem cells per se exhibit significantly greater invasive capacity, aberrant microenvironmental conditions such as hypoxia may dramatically enhance the ability of non-stem cells to invade. Consequently, both the stem and non-stem cell subpopulations of tumors may be potential targets for the development of suitable therapeutic interventions designed to inhibit prostate cancer cell invasion.

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 485. doi:10.1158/1538-7445.AM2011-485
Abstract 4863: Pathway-focused cancer mutation profiling with somatic mutation PCR arrays
Authors: Long Samuel , You Min , Wang Liang , Jiang Xiaofang , Cheng Jie , Wang Yexun
Published: June 2012

Recent advances in sequencing technologies have led to significant increases in the number of known cancer somatic mutations. Translating such information into therapeutic benefits requires molecular characterization of all major human tumor types and the advent of research tools that enable interrogation of multiple mutations in multiple cancer genes simultaneously, with high accuracy and scalability. As an increasing number of important signal transduction pathways, such as the EGFR-RAS-RAF pathway, are implicated in a variety of cancers, the utility of pathway-focused mutation analysis arrays has increased. This study examined the use of real-time PCR somatic mutation detection arrays that target all known functionally important mutation sites within an entire signal transduction pathway. Each array contains 80 to 90 ARMS® primers and 5’ hydrolysis probe-based qPCR assays that allow simultaneous detection of the most frequent and functionally important mutations for multiple genes in a specific pathway.

In this study, different cancer sample types (fresh-frozen or FFPE samples from cancer cell lines and tissues) were profiled on qPCR arrays. To establish methodology validity, pyrosequencing was used as a performance benchmark. FFPE tissue sections of lung adenocarcinoma originating from different sources and with dramatically different DNA quality were tested to examine the tolerance of qPCR-based mutation detection to sample quality variation. Finally, data analysis algorithms were developed to maximize the accuracy of mutation/genotype calls.

Results indicated that allele-specific PCR amplification combined with probe detection can achieve sufficient analytical specificity and sensitivity for detecting as little as 1% mutant alleles in a background of wild type genomic DNA. In addition, the method can detect mutations in FFPE samples over a wide range of quality (i.e. with differing extents of DNA degradation), and the mutations detected were verified by pyrosequencing analysis. Interestingly, multiple mutations across different genes within the EGFR pathway, and more than one mutation within the EGFR gene, were detected in lung adenocarcinoma samples, indicating either acquisition of multiple mutations by the same cell population or sample heterogeneity.

In conclusion, it was demonstrated that somatic mutation profiling in the context of a pathway can be easily accomplished by using qPCR arrays and can be applied to biomarker development for a variety of human cancers.

Disclaimer: The applications presented here are for research use only. Not for use in diagnostic procedures.

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 4863. doi:10.1158/1538-7445.AM2011-4863
Abstract 5123: A platform for examining the molecular basis for targeted therapy selection in colon cancer
Authors: Chen Yi , Wood Elizabeth R. , Chen Yian A. , Coppola Domenico , Siegel Erin , Shibata David , Koomen John M.
Published: June 2012

Introduction: The quantitative evaluation of expression and post-translational modification of receptor tyrosine kinase targets in tumor tissues may enable the development of a molecular profile for the selection of targeted therapy in colon cancer.

Experimental Procedures: Colon cancer cell lines are used to develop peptide-based quantitative assays for receptor tyrosine kinase protein expression and post-translational modification using SDS-PAGE protein fractionation prior to in-gel digestions and liquid chromatography coupled to multiple reaction monitoring mass spectrometry (LC-MRM). Additional downstream signaling proteins are also investigated. The sensitivity of each assay is tested using serial dilutions of cell lysate before translation to individual frozen tissue sections, which are bookmatched to hematoxylin and eosin stained slides for histology. A classification scheme for selection of targeted therapy is devised based on the amounts of protein expression and modification detected by LC-MRM.

Data Summary: LC-MRM assays have been previously developed to monitor components of the beta catenin signaling pathway have been translated to measure protein expression in individual frozen tissue sections. Using the same methodology, LC-MRM assays have been developed for receptor tyrosine kinases (RTKs), including EGFR, Met, VEGFR, and IGF1R, as well as selected RTK interacting proteins and downstream signaling components. Implementing these novel assays in parallel with those already existing, an analysis of multiple signaling pathways in frozen section of colon tumors is conducted to enable a view of the tumor biology and assess the relevance of treatment with RTK inhibitors. Conclusions: The ability to examine cancer-related pathways in human tumors with quantitative mass spectrometry will produce improved understanding of the processes involved in tumor development and progression as well as elucidate the mechanisms of cancer cell response to chemotherapy. The coupling of quantitative proteomics with medical history and histology for comprehensive analysis of tumor tissues represents a promising step toward the ultimate goal of establishing a molecular basis for personalized medicine. This effort will demonstrate the feasibility of creating a quantitative proteomics method for tumor evaluation that could be used to translate basic science hypotheses into clinical samples and build a systems biology tool for patient assessment.

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 5123. doi:10.1158/1538-7445.AM2011-5123
Abstract 5204: PP2A- STAT3 pathway mediates suspension survival in colon cancer stem cells
Authors: Chang Chia Chien , Wang Jir-You , Hung Shih-Chieh
Published: June 2012

Cancer stem or initiating cells (CSCs) which share similarity with normal stem cells are a small population of cells in tumor with the abilities of self-renewal, multi- potent differentiation, and tumor initiation. CSCs also contribute to the resistance of tumor to chemotherapy or irradiation therapy. Therefore, targeting CSCs could be a hopeful way in treating cancer.

Involvement of constitutive phosphorylation of STAT3 at Tyr705 in tumorigenesis of CSCs was implicated in many types of cancers. However, the role of Ser727 phosphorylation in STAT3 activation and CSCs tumorigenesis remains unknown. In this study, we are trying to figure out whether Ser727 phosphorylation of STAT3 plays a role in the characteristic properties of colon CSCs. Efforts will also be made to shed light on the upper signaling pathway and downstream targets of Ser727 phosphorylation of STAT3.

Our preliminary data demonstrate enriched CSCs increase in the capacity for suspension survival both in vitro and in vivo. After enrichment of CSC population by spheroid culture, the phosphorylation of STAT3 at Ser727 but not Tyr705 was increased and localized at nucleus. The increase of Ser727 activation is also associated with down-regulation of PP2A activity, a serine/threonine phosphatase candidate of STAT3. We further proved PP2A formed immunoprecipitated complex with STAT3 and regulated STAT3 activation at Ser727 by treating the cells with PP2A inhibitor in normal culture or PP2A activator in spheroid culture. Overexpression of wild-type PP2A or Ser727 point mutated STAT3 reduced the survival ability in CSC. In contrast, knock down of PP2A activity or gain of function mutation at Ser727 of STAT3 increased the survival ability in nCSC. Taken together, our current data indicate enriched CSCs increased the capacity of suspension survival through the activation of PP2A-STAT3 pathway.

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 5204. doi:10.1158/1538-7445.AM2011-5204
Abstract 539: The expression of monocyte/macrophage-restricted scavenger receptor CD163 by malignant cells may be a consequence of cell fusion with tumor-associated macrophages: A novel target for cancer therapy
Authors: Maniecki Maciej B. , Damsky William E. , Ulhøi Benedicte P. , Steiniche Torben , Ørntoft Torben F. , Dyrskjøt Lars , Borre Michael , Kjeldsen Eigil , Bosenberg Marcus W. , Møller Holger J.
Published: June 2012

Solid tumors contain large a number of infiltrating macrophages, which are believed to play a key role in progression and metastasis. Recent research has indicated that malignant cells at times express molecular pathways normally only observed in myeloid cells such as macrophages, hence suggesting that macrophages may fuse with tumor cells. In the present work, we aimed to investigate tumor cell expression of the monocyte/macrophage-restricted scavenger receptor CD163. Initially, the tumor expression of CD163 was examined using a bladder cancer tissue microarray (TMA) containing 425 cores of tumor tissue from 425 patients and 43 paired metastatic lymph nodes. Tumor cells showed a characteristic pleomorphic and atypical morphology with large nuclei, and they were easily distinguished from macrophages. Immunohistochemical staining revealed that CD163-expressing tumor cells could be detected in 98% of the tumor cores, and CD163-expressing metastatic cells could be identified in 77% of the cores of lymph node biopsies. Quantitative real-time polymerase chain reaction and flow cytometric analysis showed that bladder cancer cell lines did not express CD163; however, when co-cultured with macrophages a significant induction of CD163 expression was observed. Utilizing the xCELLigence Real-Time Cell Analyzer System we showed that macrophage-tumor hybrid cells exhibited a significantly increased migratory capacity compared to non-hybrid tumor cells. Tracing the Y chromosome in co-cultures of male macrophages and female T24 cells, we found T24 cell nuclei to be fused with the macrophages. A Cre-inducible mouse model of melanoma expressing a fluorescent reporter system (GFP) was used to characterize hybrid cell formation of macrophages with GFP positive tumors cells in vivo. Flow cytometric analysis demonstrated that >90% of the murine GFP positive tumor cells co-expressed CD163.

In conclusion, we show that the expression of the monocyte/macrophage scavenger receptor CD163 was not confined to tumor-associated macrophages. In fact, a significant portion of the tumor cells also expressed CD163, possibly as a result of heterotypic cell fusion between tumor cells and macrophages. Our findings identify CD163 as a possible target for novel cancer therapy.

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 539. doi:10.1158/1538-7445.AM2011-539
Abstract 5443: A novel biosensor for monitoring intracellular cGMP in live cells
Authors: Sun Yanjie , Keeton Adam B. , Tinsley Heather N. , Butler Braeden L. , Binkowski Brock F. , Piazza Gary A.
Published: June 2012

The second messenger, cyclic guanosine 3’, 5’-monophosphate (cGMP) is a critical and multifunctional signaling molecule involved in many cellular processes. Intracellular cGMP is synthesized by guanylyl cyclase (GC) and is degraded by various phosphodiesterase (PDE) isozymes. Increased levels of cGMP can activate cGMP-dependent protein kinase (PKG), modulate cGMP-gated ion channels, or affect PDE activity. Since cGMP plays an important physiological role and may also be involved in various pathological conditions, including cancer, CNS disorders, and vascular deficiencies, cGMP-specific PDE isozymes represent an important class of drug targets. In order to measure intracellular cGMP levels in live cells, we constructed a novel genetically encoded cGMP biosensor with cGMP binding domains (from human PDE5A GAF-A domain) fused to a modified firefly luciferase. A lipid based reagent was used to transiently transfect HEK293 with this biosensor. Cells were then treated with the nitric oxide donors, sodium nitroprusside (SNP) or NOR-3, which are known to activate soluble GC. Bioluminescence was measured every 2 minutes for at least 60 minutes following treatment. Both SNP (50 µM) and NOR-3 (100 µM) caused a 3-fold increase in bioluminescence compared with vehicle treated cells, reaching a maximum within 10 to 20 minutes of compound addition. Co-transfection with soluble GC further increased the response up to 13-fold following SNP treatment. In addition, PDE inhibitors such as, zaprinast, dipyridamole and 3-isobutyl-1-methylxanthine amplified the response to SNP treatment. As expected, co-transfection with PDE5A decreased the basal level of cGMP and increased the sensitivity of the cGMP response to zaprinast and SNP, as well as the combination. These results suggest that this cGMP biosensor can be useful for studying drug effects on cGMP signaling in live cells. Methods to use the assay for high throughput screening are also being developed. Funding provided by NIH/NCI grants CA131378 and CA148817.

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 5443. doi:10.1158/1538-7445.AM2011-5443
Abstract 5568: Ginkgo biloba extract kaempferol induces apoptosis in human prostate cancer cells through downregulation of β-Catenin signaling
Authors: Zeng Yan , Xiao Dong
Published: June 2012

Kaempferol (Kae) is one of the most important constituents in ginkgo biloba extract which has been well known for its antioxidant and anti-inflammatory activities. Kae is also reported to suppress the growth of cancer cells but the mechanism of cell death induction is not fully understood. In the present study, we performed in vitro studies, for the first time to examine whether Kae induces apoptotic cell death in human prostate cancer cells and if β-Catenin signaling pathway is involved in the Kae-induced apoptosis. Kae treatment was found to decrease viability of human prostate cancer PC-3 (androgen independent, p53 null) and LNCaP (androgen responsive, wild-type p53) cells at pharmacologically achievable concentrations (10-40 µmol/L at 24 h). The anti-cancer activity by Kae was caused by apoptosis induction, which was determined by increased cytoplasmic histone-associated DNA fragmentation, caspase 3 activities and sub-G0/G1 cell phase in PC-3 and LNCaP cells treated with Kae compared with controls. Interestingly, a normal human prostate epithelial cell line (PrEC) is significantly more resistant to growth inhibition and apoptosis induction by Kae. Exposure of PC-3 and LNCaP cells to Kae resulted in significant inhibition of β-Catenin protein levels by Elisa assay, down-regulation of protein expression of β-Catenin as well as its down stream targets C-myc, Cyclin D1 and survivin, and alteration in nuclear/cytoplasmic distribution of β-Catenin by immunoblotting. Moreover, Kae treatment significantly inhibited the β-Catenin/TCF transcriptional activity in PC-3 and LNCaP cells transiently transfected with the TCF-4 reporter assay. We also found that apoptosis induction by Kae in human prostate cancer cells correlates with suppression of androgen receptor (AR), phosphor-AR and PSA protein levels. In conclusion, the present study indicates that β-Catenin as well as AR signaling pathways may be targets for Kae-induced apoptosis in human prostate cancer cells. This investigation was supported by USPHS grant R21 CA143104-02, 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 5568. doi:10.1158/1538-7445.AM2011-5568
Abstract 819: Black raspberries inhibit N-nitrosomethylbenzylamine (NMBA)-induced rat esophageal tumorigenesis through down-regulation of MAPK and NFκB pathways
Authors: Yan Fei , Guan Huaji , Wang Yufen , Battista Carter , Huang Xin , Nines Ronald G. , Edmonds Alexandria M. , Stoner Gary D. , Chen Tong
Published: June 2012

Esophageal cancer is the 6th most common cancer worldwide. Our laboratory has used a rodent preclinical model of esophageal squamous cell carcinoma to identify putative chemopreventive agents for this disease and to determine their mechanisms of action. In our previous studies, we found that freeze-dried black raspberry (BRB) significantly suppressed N-nitrosomethylbenzylamine (NMBA)-induced tumor development in rat esophagus, at least in part by inhibition of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), c-Jun and vascular endothelial growth factor (VEGF). The mitogen-activated protein kinase (MAPK) and nuclear factor κB (NFκB) pathways play important roles in certain aspects of carcinogenesis. However, their roles and effects of BRB on these two pathways in the NMBA model of rat esophageal tumorigenesis have not been investigated yet. In the present study, F344 rats were treated with NMBA (0.30 mg/kg b.w.) three times per week for 5 weeks. After 72 hours, animals were fed AIN-76A diet or AIN-76A diet containing 5% BRB. At week 35, rats were sacrificed and esophageal tumors were counted. The incidence of esophageal tumors was decreased from 100% in NMBA-treated rats to 81.48% (P < 0.05) in rats treated with NMBA+5% BRB. The tumor multiplicity was reduced in rats fed 5% BRB to an average of 1.44 ± 0.25 (P < 0.001) tumors per rat compared with 4.73 ± 2.70 in rats fed the control diet. In preneoplastic lesions, BRB decreased protein expression of phospho-NFκB-p65, phospho-IkBα, phospho-MAPK-p38, phospho-MAPK-p44/42 and phosphor-SAPK/JNK from 4.2-fold, 3.9-fold, 3.1-fold, 4.7-fold and 5.5-fold in rats fed the control diet to 1.2-fold (P < 0.005), 1.3-fold (P < 0.005), 1.3-fold (P < 0.005), 1.4-fold (P < 0.005) and 1.4-fold (P < 0.005), respectively, in rats fed 5% BRB. In papillomatous lesions, BRB reduced protein expression of phospho-NFκB-p65, phospho-IkBα, phospho-MAPK-p38, phospho-MAPK-p44/42 and phosphor-SAPK/JNK from 5.9-fold, 4.7-fold, 5.7-fold, 5.9-fold and 6.1-fold in rats fed the control diet to 1.3-fold (P < 0.005), 1.5-fold (P < 0.005), 1.6-fold (P < 0.005), 1.5-fold (P < 0.005) and 1.6-fold (P < 0.005), respectively, in rats fed 5% BRB. Our results indicated that both MAPK and NFκB pathways were activated in NMBA-induced tumor development in rats and these activations were significantly inhibited by BRB. Collectively, our data suggest that BRB may offer a relatively nontoxic alternative to the prevention of esophageal cancer in humans. (Supported by NCI RO1 CA131073-01A1).

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 819. doi:10.1158/1538-7445.AM2011-819
Abstract 831: Chemopreventive effects of selective estrogen receptor modulator (SERM) raloxifene on intestinal neoplasia of APCMin/+ mice
Authors: Janakiram Naveena B. , Mohammed Altaf , Qian Li , Venkateshwar Madka , Reddy Manasa P. , Steele Vernon E. , Rao Chinthalapally V.
Published: June 2012

Estrogen receptor β (ER-β) has been implicated in several neoplastic diseases. Previously we have shown that ER-β is up-regulated in human and rodent colonic tumors, suggesting ER-β as a potential target for colon cancer prevention (Naveena et al., CaPR 2009). Thus, ER-β modualtors, SERMs are useful for colon cancer prevention. Hence, we tested raloxifene, an ER antagonist in APCMin/+ mice treated with azoxymethane (AOM), to enhance the colonic tumors. Six-week-old male and female APCMin/+ (9/group) were injected s.c., with AOM at 5 mg/kg BW, once a week for three weeks. APCMin/+ mice were then fed diets containing 0, 1.5, and 3.0 ppm raloxifene for 12 weeks before intestinal tumors and various markers of toxicity, cell proliferation and apoptosis were evaluated. Results suggest that dietary administration of 1.5 and 3.0 ppm raloxifene in male mice suppressed small intestinal polyp formation by 24% (p<0.05) and 58% (p<0.0001), respectively. In female mice, 1.5 and 3.0 ppm raloxifene suppressed small intestinal polyps by 58% (0<0.0001) and 48% (p<0.0001), respectively. Also, significant inhibition of colonic tumor suppression was observed in female mice fed the 1.5 and 3.0 ppm of raloxifene, whereas in male mice, only high-dose inhibited colonic tumors. Mice fed with raloxifene had >70% less polyps with size of >1-mm suggesting suppression of tumor volume/size. Also, we find that raloxifene treatment in female APCMin/+ mice showed significantly reduced serum creatine phosphokinase and cholesterol levels, and an increased alkaline phosphatase levels when compared to untreated control mice. Also, a dose-dependent increase in serum calcium was observed with raloxifene administration. Dietary raloxifene showed no liver enzyme toxicities as measured by aspartate aminotransferase levels in serum. However, raloxifene administered mice showed a modest decrease in serum glucose levels. The mRNA and protein expression levels for p21cip/waf1 were significantly higher with decreased expression of Cyclin D1 and PCNA in raloxifene treated colonic tumors and SI polyps compared to control diet fed mice tissues, suggesting estrogen's function in crypt cell proliferation. Overall, our results clearly suggest that raloxifene significantly suppresses small intestinal and colonic polyps formation by up-regulating p21cip/waf1 and has potential usefulness for human colon cancer prevention. (Supported by CN53300 and R01-94962)

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 831. doi:10.1158/1538-7445.AM2011-831
Abstract 832: Effects of beta-cryptoxanthin on cigarette smoke-induced lung oxidative damage, inflammation and activation of NF-kappa B and AP-1
Authors: Liu Chun , Bronson Roderick T. , Russell Robert M. , Wang Xiang-Dong
Published: June 2012

In epidemiologic studies, high intake of beta-cryptoxanthin has been associated with a decreased risk of lung cancer, particularly among current smokers. However, data are not available from well-controlled animal studies to examine the effects of beta-cryptoxanthin on cigarette smoke-induced lung lesions, and the biological mechanisms by which beta-cryptoxanthin might affect lung carcinogenesis. We evaluated the effects of beta-cryptoxanthin supplementation on cigarette smoke-induced squamous metaplasia, inflammation, and changes in protein levels of pro-inflammatory cytokine [tumor necrosis factor alpha (TNF-alpha)] and transcription factors [nuclear factor kappa B (NF-kappa B) and activator protein-1 (AP-1)], as well as on smoke-induced oxidative DNA damage [8-hydroxy-2′-deoxyguanosine (8-OHdG)] in the lung tissue of ferrets. Thirty six male ferrets were assigned to cigarette smoke exposure or to no exposure and to no dose, low-dose, or high-dose beta-cryptoxanthin supplementation (2 × 3 factorial design) for 3 months. Beta-cryptoxanthin supplementation dose-dependently increased plasma and lung beta-cryptoxanthin levels in ferrets, whereas cigarette smoke exposure lowered plasma and lung beta-cryptoxanthin levels. Beta-cryptoxanthin at both doses significantly decreased smoke-induced lung squamous metaplasia and inflammation. Beta-cryptoxanthin also substantially reduced smoke-elevated TNF-alpha levels in alveolar, bronchial, bronchiolar and airway serous/mucous gland epithelial cells and in lung macrophages. Moreover, beta-cryptoxanthin decreased smoke-induced activation of NF-kappa B and expression of AP-1 and 8-OHdG. The beneficial effects of beta-cryptoxanthin were stronger for high-dose beta-cryptoxanthin than for low-dose beta-cryptoxanthin. Data from this study indicate that beta-cryptoxanthin provides a beneficial effect against cigarette smoke-induced inflammation, oxidative DNA damage and squamous metaplasia in the lungs. Beta-cryptoxanthin may prevent lung carcinogenesis through its inhibitive effects on lung inflammation and oxidative DNA damage.

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 832. doi:10.1158/1538-7445.AM2011-832
Abstract 894: Pancreatic cancer screening by TLR phenotyping
Authors: Rosenberg Jonathan S. , Singh Sujay , Raul Sanjay , Tong Zhimin , Guha Sushovan
Published: June 2012

Background: Toll-like receptor (TLR) proteins play key roles in immune responses against infection. TLRs are normally associated with multiple chronic inflammatory diseases. As chronic inflammation often leads to development of neoplasia, we hypothesize that TLRs may promote neoplasia in distinct organ types. Elevated expression of some TLRs has been reported in many tumor tissues and/or tumor cell lines. It is known that in pancreatic ductal adenocarcinoma (PDAC), chronic inflammatory conditions often precede the tumorigenesis process. However, in this context a comprehensive analysis of expression of TLRs in PDAC is currently unknown.

Aim: To investigate the expression of multiple TLRs in PDAC cells.

Methods and Results: Multiple PDAC cell lines including AsPC-1, MIA PaCa2, PANC-1, PANC-28, and SW1990 were used for TLR phenotyping. The expression of TLRs including TLR2, 3, 4, 7 and 9 were analyzed by Flow Cytometry and Western Blotting techniques. We found that the expression of TLR2, 3, 4, 7 and 9 can be detected in MIA PaCa2, PANC-1, PANC-28, and SW1990 cells. However, the expression of TLR4 and TLR7 are very low in SW1990 cells when compared to MIA PaCa2, PANC-1, and PANC-28 cells. In AsPC-1 cells, the expression of TLR2, 4, and 7 are below the detection levels while the expression of TLR9 and TLR3 can be readily detected.

Conclusion: The expression profile of TLRs is distinct among multiple PDAC cells. Using the PDAC cell lines as a model system we are assessing potential predictive value of TLRs as biomarkers in cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 894. doi:10.1158/1538-7445.AM2011-894
Abstract 92: Effects of p16 methylation on the efficacy of cetuximab in metastatic colorectal cancer
Authors: Kim Ki Hyang , Park Kyu Hyun , Chang Hyun , Kim Hyo Song , Shin Sang Joon , Rha Sun Young , Chung Hyun Cheol , Roh Jae Kyung , Ahn Joong Bae
Published: June 2012

Background: Promoter DNA methylation is an important epigenetic factor of gene inactivation in human carcinogenesis and gene silencing of tumor suppressor genes in colorectal cancer. Global DNA methylation, reflected by the status of LINE-1 methylation, plays an important role in genomic instability and carcinogenesis. We examined the role of p16 methylation and LINE-1 methylation in metastatic colorectal cancer patients treated with cetuximab chemotherapy.

Methods: The study was performed using fresh frozen specimens from 42 metastatic colorectal cancer patients treated with cetuximab. Samples were analyzed by bisulfied-based approaches to study the methylation status of p16 and LINE-1 by pyrosequecing. KRAS mutation was determined by pyrosequencing.

Results: Methylation of p16 was associated with decreased progression-free survival in all patients (n=42, 4.9 months in p16 methylated patients vs 15.7 months in p16 unmethylated patients, p=0.01). When proximal location tumors were analyzed separately, methylation of p16 was associated with decreased progression-free survival (n=11, 4 months vs 15.7 months, p<0.01), and decreased overall survival (14.5 months vs 35 months, p<0.01). Among KRAS wild type tumors (n=35), methylation of p16 demonstrated a tendency toward decreased progression-free survival (6.3 months in methylated patients vs 15.7 months in unmethylated patients, p=0.07). In 3rd line erbitux treatment patients (n=13), the median PFS was 4.0 months for p16 methylated patients and 9.6 months for unmethylated patients (p=0.25). In the response rate analysis, compared with KRAS wild type tumors, KRAS mutant tumors had a significantly lower response rate (0 % vs 45.7%, p=0.033). Compared with P16-methylated tumors, p16 unmethylated tumors demonstrated a tendency toward better response rate (10% vs 46.9%, p=0.061). LINE-1 methylation was not associated with either progression-free survival (p=0.53) or response rate (p = 0.53).

Conclusions: 16 methylation may be a factor other than KRAS mutation associated with resistance to anti-EGFR monoclonal antibodies. Because we analyzed heterogeneous population and our sample size was too small, we suggest that further studies are necessary to determine the predictive role of DNA methylation 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 92. doi:10.1158/1538-7445.AM2011-92
Abstract 924: Identification of somatic mutations of the PPP2R1A gene in subtypes of endometrial and ovarian carcinomas
Authors: McConechy Melissa K. , Anglesio Michael S. , Kalloger Steve E. , Yang Winnie , Senz Janine , McAlpine Jessica , Gilks Blake , Huntsman David
Published: June 2012

Somatic mutations in PPP2R1A have been found at low frequencies in lung, breast cancer and melanoma and most recently through whole-exome sequencing in ovarian clear cell carcinoma. We examined whole-transcriptome sequencing data from ovarian clear cell carcinomas (OCCC) and identified a cluster of mutations in exon 5 of PPP2R1A. The protein phosphatase 2, regulatory subunit A, alpha (PPP2R1A, or PR65a) gene is the scaffolding subunit of the serine-threonine protein phosphatase 2A (PP2A) holoenzyme. PP2A is involved in growth and survival cellular pathways, and has been suggested to play a role as a tumor suppressor gene in some human cancers. The PP2A holoenzyme is made up of the A scaffolding subunit (isoforms α and β), the C catalytic subunit (isoforms α and β and multiple regulatory B subunits also with many isoforms. PPP2R1A is made up of 15 HEAT domains forming a horseshoe-like multi-helical structure that interacts with both the C and B subunits of PP2A. We have examined the frequency of PPP2R1A mutations across the spectrum of both ovarian and endometrial carcinomas. DNA from endometrial and ovarian subtypes was sequenced across exons 5 and 6 of PPP2R1A using PCR based Sanger sequencing, and somatic mutation status was verified by sequencing matched normal DNA. Targeted sequencing of PPP2R1A revealed somatic missense mutations in 36.7% (18 of 49) of endometrial high-grade serous primary tumors. This high frequency of mutations has not previously been reported and suggests a strong link to the pathology of this disease subtype. Mutations were also identified at a lower frequency of 4.9% (3 of 61) in endometrial endometrioid carcinomas, and ovarian primary tumors at 11.6% (5 of 43) in endometrioid, 4% (2 of 50) in clear cell, 0% (0 of 50) in high-grade serous and 0% (0 of 12) of low-grade serous carcinomas. These nucleotide missense mutations result in the modification of amino acid residues that are highly conserved within the intrarepeat helix of the HEAT domains of PPP2R1A. Previous mutation based biochemical studies have identified these amino acid residues as important in the interaction between the regulatory B subunit and PPP2R1A. This indicates a strong functional role of PPP2R1A in stabilization of the PP2A complex, and a probable role in cellular tumorgenesis. A closer survey of mutations found within PPP2R1A and the PP2A complex, as well as functional characterization of this pathway, may pave the way to defining biologically relevant pathways and targets within endometrial carcinomas, ultimately leading to improved patient-targeted therapies and survival.

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 924. doi:10.1158/1538-7445.AM2011-924
Abstract 993: Enhanced EGFR activity of prostate cancer cells by low pH, low glucose environment
Authors: Bollu Lakshmi R. , Weihua Zhang
Published: June 2012

Background: Hypoxia, lack of nutrients and high aerobic glycolysis creates a microenvironment of high lactate, low glucose and low pH conditions within a solid tumor. The purpose of the current research is to study how the cancer cells adapt to a microenvironment with low pH, low glucose and high lactate levels with selective growth advantages.

Methods: We simulated solid tumor microenvironment conditions by making DMEM media with low glucose (2mM), high lactate (10mM) and low pH (6.5). To study the growth advantages of this microenvironment, we determined the effect of low pH, low glucose and lactate medium on EGFR activation. Prostate cancer (PC3) cells were grown in six-well plates in 10% serum containing DMEM and serum starved overnight before they were changed to experimental medium. Control cells were grown in normal DMEM. We treated PC3 cells growing in normal DMEM and experimental medium with EGF at 1ng/ml. Protein samples were collected at different time intervals and subjected to western blotting to observe the activation of EGFR by using phospho EGFR antibody. EGFR and actin antibodies were used as control and loading controls, respectively.

Results: Robust and sustained phosphorylation of EGFR for longer time was observed with EGF treatment when the cells were grown in low pH, low glucose and high lactate medium when compared with the cells grown in normal DMEM medium.

Conclusion: The robust activation of EGFR with low pH, low glucose and high lactate medium suggest that the microenvironment containing high lactate provides growth or survival advantages to the 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 993. doi:10.1158/1538-7445.AM2011-993
Abstract LB-12: Estrogen-induced redox signaling via NRF-1 is involved in the development of breast cancer
Authors: Okoh Victor , Felty Quentin H. , Roy Deodutta
Published: June 2012

The purpose of this study was to determine the role of estrogen-induced reactive oxygen species (ROS) signaling pathways in the development of breast cancer. Here we tested the postulate that activation of a redox sensitive transcription factor, nuclear respiratory factor-1 (NRF-1) signaling is essential for 4-hydroxyestradiol (4-OH-E2)-induced tumorigenesis. We observed that (i) 4-OH-E2 treatment of MCF-10A or MCF-7 cells increased the formation of ROS, (ii) Estrogen-induced ROS activated redox sensitive transcription factor NRF-1, (iii) 4-OH-E2 through activating a redox sensitive serine-threonine kinase AKT phosphorylated NRF-1, (iv) Overexpression of catalase and MnSOD, as well as silencing of NRF-1 prevented 4-OH-E2-induced anchorage-independent growth of MCF-10A and MCF-7 cells and xenograft tumor formation, and (v) 4-OH-E2-induced ROS through stimulating AKT kinase and histone acetyltransferase of CREB binding protein-associated factor (P/CAF), respectively, controlled phosphorylation and acetylation of NRF-1. The results also showed that 4-OH-E2-induced post-translational changes lead to NRF-1 activation, and silencing of NRF-1 and overexpression of MnSOD or catalase prevented 4-OH-E2-induced tumor formation. Taken together, these findings suggest a new paradigm for estrogen-induced breast carcinogenesis that deregulation of phosphorylation and aceytylation of NRF-1 may represent a major mechanism of 4-OH-E2-induced neoplastic transformation and tumorigenesis.

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 LB-12. doi:10.1158/1538-7445.AM2011-LB-12
Abstract LB-125: Germline mutations in BAP1 predispose to melanocytic nevi and melanoma
Authors: Wiesner Thomas , Obenauf Anna , Murali Rajmohan , Fried Isabella , Griewank Klaus , Ulz Peter , Windpassinger Christian , Loy Shea , Wackernagel Werner , Wolf Ingrid , Becker Juergen , Viale Agnes , Lash Alex , Pirun Mono , Socci Nick , Ruetten Arno , Palmedo Gabrielle , Ott Arthur , Abramson David , Offit Kenneth , Cerroni Lorenzo , Kutzner Heinz , Bastian Boris , Speicher Michael
Published: June 2012

We describe an autosomal-dominant syndrome characterized by multiple non-pigmented, exophytic melanocytic nevi and an increased susceptibility for melanoma, caused by germline mutations in the histone deubiquitinase BAP1. To identify the causative alterations, we performed comprehensive genomic analyses in two unrelated families with numerous dermal nevi composed largely of large, epithelioid melanocytes with abundant amphophilic cytoplasm and large, pleomorphic, vesicular nuclei with prominent nucleoli. Both families each had one proband with uveal melanoma, and three probands in one family had cutaneous melanoma. Array-based comparative genomic hybridization (aCGH) revealed losses of parts of or the entire chromosome 3 in 11 of 22 neoplasms studied. Genotypic analyses revealed that the deletions invariably affected the chromosome from the unaffected parent. Genome partitioning of the minimally deleted region on chromosome 3p21 followed by massively parallel sequencing revealed two different inactivating germline mutations of the BAP1 tumor suppressor gene that in both families segregated with the phenotype. In almost all tumors the remaining wild type BAP1 allele was eliminated by deletion, separate inactivating mutations, or loss of heterozygosity. 35 of 40 nevi (88%) showed mutations in BRAF, while the uveal melanomas had mutations in GNAQ.

Our data identify BAP1 as a highly penetrant susceptibility gene for melanocytic neoplasia. Somatic BAP1 mutations have recently been reported in uveal melanoma and linked to the metastatic phenotype. Our observation of frequent bi-allelic inactivation of BAP1 in nevi indicates that the role of BAP1 in melanocytic neoplasia is more complex, and may differ depending on other factors such as the type of melanocyte (uveal or cutaneous) and the co-existing oncogenic mutation.

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 LB-125. doi:10.1158/1538-7445.AM2011-LB-125
Abstract LB-15: Overexpression of Pak1 and Pak4 contributes to endometrial carcinogenesis
Authors: Siu Michelle KY , Kong Daniel SH , Chan Hoi Yan , Ngai Sheila YP , Jiang Lili , Wong Esther SY , Ngan Hextan YS , Cheung Annie NY
Published: June 2012

Introduction: Endometrial cancer is the most common gynaecological cancer worldwide and its incidence in Asia is rising. It can be classified into two major types: the estrogen dependent type I endometrioid carcinoma that comprises about 80% of endometrial cancers and the type II non-endometroid carcinoma, which is estrogen-independent, including serous carcinoma and clear cell carcinoma. p21-activated kinases (Paks) are major effectors of the small Rho GTPases Rac1 and Cdc42 that play important roles in cell morphology, cytoskeletal reorganization, apoptosis, survival and angiogenesis. This study investigated the expression patterns, clinical significance, functional roles and putative downstream targets of Pak1 and Pak4 in endometrial cancer. Methods and Results: Immunohistochemical study of Pak1, Pak4 and p-Pak4 Ser474 (the activated form) was performed on 10 normal atrophic endometrium, 16 hyperplastic endometrium without atypia, 17 atypical complex hyperplasia and 67 endometrial carcinomas. Pak1 was expressed in cytoplasm whereas Pak4 and p-Pak4 were expressed in both cytoplasm and nucleus. Significantly higher Pak1, Pak4 and p-Pak4 expression was detected in hyperplastic endometrium and endometrial carcinomas than normal atrophic endometrium. Moreover, endometrioid carcinomas showed significantly higher Pak1 expression than non-endometrioid carcinomas. In endometrial carcinomas, expression of Pak4 was found to be correlated with proliferation markers, MCM7 and Ki67. By quantitative real-time polymerase chain reaction, significantly higher Pak1 and Pak4 mRNA expression was observed in endometrial cancer samples when compared with the corresponding non-tumor counterparts. Up-regulation of Pak1 and Pak4 mRNA and protein expression was also found in cancer cell lines compared with normal endometrial cells. Knockdown of Pak1 by siRNA in RL-95–2, an endometrial cancer cell line with relatively high Pak1 expression, inhibited cell proliferation along with down-regulation of estrogen receptor alpha mRNA and protein expression. Conclusion: This is the first report revealing the expression of Pak1 and Pak4 in normal atrophic, hyperplastic endometrium and endometrial carcinomas, suggesting Pak1 and Pak4 to be useful markers in detection of endometrial carcinoma in postmenopausal women and potential therapeutic targets in endometrial cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-15. doi:10.1158/1538-7445.AM2011-LB-15