bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract A34: A mechanism for acquired resistance to estrogen deprivation in breast cancer cells
Authors: Aguilar Helena , Bonifaci Nuria , Sole Xavier , Musach Jordi Serra , Moreno Victor , Lazaro Conxi , Urruticoechea Ander , Pujana Miguel Angel
Published: January 2011

Endocrine therapies that target the proliferative effect of 17β-estradiol (17βE2) through estrogen receptor α (ERα) are the most effective form of systemic treatment of ERα-positive breast cancer. Nevertheless, through mechanisms that are not fully understood, most tumors that initially respond to therapy will recur. The long-term estrogen deprived (LTED) MCF7 cell model has been proposed to recapitulate acquired resistance to aromatase inhibition (AI) in postmenopausal women. To comprehensively elucidate this resistance, genomic, transcriptomic and molecular analyses were integrated into the time course of MCF7-LTED adaptation. Extensive and dynamic genomic changes were observed, including amplification of the ESR1 locus linked to an increase in ERα and its non-genomic role. Dynamic transcriptomic profiles were also observed that significantly correlated with genomic changes and that were influenced by transcription factors known to be involved in acquired resistance (IRF-1) and cell proliferation (E2F1) but not by canonical ERα genomic function. Evaluation of MCF7-LTED profiles association with pathological and clinical data shown consistent correlations with ERα-negative tumor status, letrozole effects and recurrence after tamoxifen treatment. This study describes the coordinated genomic, transcriptomic and molecular changes that mediate acquired resistance to endocrine therapies, suggesting a key role for the non-genomic function of ERα.

Citation Information: Clin Cancer Res 2010;16(7 Suppl):A34
Abstract A7: Combined copy number and expression analysis of formalin fixed-paraffin embedded (FFPE) tumours
Authors: Iddawela Mahesh , Rueda Oscar , Wang Yuker , Faham Malek , El-Sheemy Mohamed , Cowley Ged , Eremin Jennifer , Eremin Oleg , Earl Helena , Caldas Carlos
Published: January 2011

Introduction: FFPE tissues are a valuable source of tumour material for translational research that has thus far been under-used. But progress in technology has allowed the analysis of genome wide copy number and expression changes using small amounts of degraded DNA/RNA extracted from FFPE tissues. We used two platforms: molecular inversion probes assay (MIP, Affymetrix) and c-DNA mediated Annealing Selection extension and Ligation assay (DASL, Illumina™) for analysis of RNA/DNA extracted from routine core biopsies from a clinical trial to identify molecular markers of response or resistance to chemotherapy.

Method: FFPE diagnostic core biopsies of 63 patients treated with neoadjuvant adriamycin/cyclophosphamide followed by taxotere in a clinical study were used. MIP assay with 50K SNP panel was used for copy number analysis (CNA). DASL assay assessed expression of 502 genes using three probes per gene.

Results: RNA and DNA of good quality were extracted from 52/63 (82%) and 46/63 (73%) core biopsy samples, respectively. Gene expression profiling was carried out using the DASL assay and there were good correlations with IHC for ER, PR and HER-2 with area under the curve of 0.95, 0.90 and 0.96 respectively, following ROC analysis for the best transcripts. Differential expression analysis between ER+ vs. ER- cancers showed that ESR1, TFF1 and LAF4 are significantly over-expressed in ER + cancers (p<0.001), while analysis of pathological complete responders vs. non responders showed over expression of CXCL9, ARHA, ARHGDIB, BCL3, ERBB2, BMYB and TGFBI among responders (p<0.2). CNA showed well described aberrations in breast cancer such as high probability of gains in 1q, 8q, 11q and 20q and loss in 4q, 12q, 13q, 18q and 22q (probability of alteration ≥0.35). Samples with pathological complete response had high probability of gains in 1q (55%), 8q (40%), 17q (40%) and losses in 8p (40%) and 13q (70%). 5/46 samples were HER-2 3+ with IHC and all showed amplification around the 17q locus.

Conclusions: Reliable copy number and expression data can be generated using these assays from FFPE core biopsy samples. Role of LAF4 as an oestrogen regulated gene and roles of CXCL9, ARHA and ARHGDIB as markers of response to chemotherapy should be further investigated.

Citation Information: Clin Cancer Res 2010;16(7 Suppl):A7
Abstract B19: Pentostatin facilitates fully MHC-disparate murine mini-transplantation
Authors: Massey Paul R. , Taylor Justin , Ryan Kaitlyn , Foley Jason , Mariotti Jacopo , Fowler Daniel H.
Published: January 2011

Reduced-intensity conditioning regimens prior to allogeneic hematopoietic stem cell transplantation (RIC allo-HSCT) contain cytotoxic agents to create marrow space for donor cells and rely on graft-vs-leukemia effect for curative potential. Pentostatin is in the same class of chemotherapeutics as the purine analog fludarabine, which is commonly used in RIC allo-HSCT. However, pentostatin has a unique mechanism of action that targets adenosine deaminase and may allow for selective targeting of lymphocytes. We hypothesize that pentostatin can be used as an alternative to fludarabine in an MHC-disparate murine setting (stringent model).

We compared the immune effects of pentostatin (1 mg/kg/d) and fludarabine (100 mg/kg/d) alone to each in combination therapy with cyclophosphamide (50 or 100 mg/kg/d). BALB/c mice were treated with a three-day regimen followed by transplantation of B6 bone marrow. Combination PC or FC were each more effective at depleting and suppressing splenic T cells than either agent alone; however, the PC regimen was synergistic in depleting host CD4+ T cells (p<0.01) and CD8+ T cells (p<0.01), and suppressing their function. Immune depletion was characterized by spleen harvest followed by flow cytometry at different times during treatment, while host immune suppression was evaluated by cytokine content in multiplex bead array (Bio-Rad).

Surprisingly, recipients in both cohorts rejected the graft. We then reasoned that the rejection was mediated by host immune reconstitution. Indeed, CD4+ T cells of BALB/c mice rebounded to 22.4% (p<0.0001) of pre-treatment levels 10 days after conditioning with PC, and cohorts treated with FC rebounded to 58.8% (p<0.001) of pre-treatment levels (means of 26.7∗106 cells in HBSS, 6.0 ∗ 106 for P, 15.7∗106 for F, 1.9∗106 for XRT).

We successfully developed a more immunodepletive regimen by intensification of cyclophosphamide therapy to 14 days with P or F administered every 3 days. In this model, PC demonstrated approximately a 75-fold decrease in CD4+ cells two weeks after the end of treatment (8.3 × 106 v. 0.12 × 106, p<0.05) and FC a 15-fold decrease (0.53 × 106, p<0.05) compared to controls; granulocytes were preserved in both cases (#Gr1+ cells). The 14-day regimen also allowed for engraftment in both PC and FC, with 93% of PC–treated and 57% of FC-treated cohorts achieving >5% donor chimerism (p<0.0001).

In conclusion, pentostatin facilitates engraftment in stringent, fully-MHC disparate model, therefore we posit it can be used as a fludarabine alternative in a reduced-intensity human transplant setting.

Citation Information: Clin Cancer Res 2010;16(7 Suppl):B19
Abstract CN2-1: Personalized chemotherapy of cancer based on immune parameters
Authors: Zitvogel Laurence , Kroemer Guido
Published: January 2011

Recent data suggest that therapy-induced immunogenic cancer cell death can stimulate a therapeutic anti-cancer immune response that then contributes to the control (or even to the elimination) of residual tumor cells. Indeed, some chemotherapeutic agents (e.g. X Rays, oxaliplatin and anthracyclines) can induce immunogenic apoptosis, while others cannot (e.g. cisplatin, mitomycin C and etoposide) [Casares, 2005; Obeid, 2007; Apetoh, 2007; Obeid, 2007; Ghiringhelli, 2009]. Thus far, we know that the immunogenicity of tumor cell death is closely linked to the surface exposure of calreticuline (CRT), the secretion of ATP and the release of HMGB1 that bind to CRT receptor, P2RX7 and TLR4 on host dendritic cells respectively. Chemotherapeutic agents that fail to induce CRT exposure or HMGB1 release are unable to induce immunogenic cell death. Our published data indicate that two loss-of-function alleles (that affect toll-like receptor-4 [TLR4] and purinergic P2 receptor X7 [P2RX7] of 12 and 30% of Caucasians, respectively) reduce the efficacy of conventional anticancer therapies, for instance in anthracyline-treated breast carcinomas and in oxaliplatin-treated colon cancers. We have determined novel defects in the emission or perception of immunogenic cell death signals negatively affecting the therapeutic response of human cancers that will be presented. Mouse preclinical models allowed us to unravel novel cellular and molecular pathways involved in the immunogenicity of cell death. These data will be reviewed.

Based on this concept, which remains to be proven, one can formulate four predictions:

First, therapeutic regimens that are intrinsically unable to elicit immunogenic death should be less efficient than therapies that can trigger immunogenic cell death. Second, cancers that intrinsically lack the capacity to emit immunogenic signals during tumor cell death should fail to induce a specific immune response and have a bad prognosis. Third, immune defects that weaken the response against dying tumor cells, including defects in the perception of immunogenic signals, should worsen the prognosis of cancer and compromise the therapeutic response. Fourth, the immunogenic cell death induced by chemotherapy should influence the composition and the architecture of the immune infiltrate present in tumors, which in turn should affect the therapeutic response.

Citation Information: Clin Cancer Res 2010;16(7 Suppl):CN2-1
Abstract ED3-3: Etiological clues from breast cancer GWAS
Authors: Garcia-Closas Montserrat
Published: January 2011

Breast cancer is a heterogeneous disease and risk factors can be differentially associated with the development of distinct tumors subtypes that manifest different biological and clinical behavior. Tumor subtypes, such as those defined by hormone receptor expression, show different age-specific incidence rate patterns and risk factor profiles. In addition, tumors from BRCA1 and BRCA2 mutation carriers display distinct pathological characteristics. Large collaborative efforts to conduct genome-wide association studies (GWAS) have lead to recent discoveries of low penetrance, common susceptibility loci that also show distinct associations by tumor subtypes. Follow up studies are evaluating the biological mechanisms underlying the observed risk associations, as well as potential gene-gene and gene-environment interactions. The relationship between genetic variants associated with pathological subtypes of breast cancer, and the implication of discoveries of novel genetic variants to risk prediction in BRCA1/2 mutation carriers and the general population will be discussed. These discoveries might lead to improvements in our understanding of the etiology of specific tumor types, which may enable the development of targeted prevention, early detection, and treatment strategies.

Citation Information: Clin Cancer Res 2010;16(7 Suppl):ED3-3
Sorafenib Inhibits the Hepatocyte Growth Factor–Mediated Epithelial Mesenchymal Transition in Hepatocellular Carcinoma
Authors: Nagai Tomoyuki , Arao Tokuzo , Furuta Kazuyuki , Sakai Kazuko , Kudo Kanae , Kaneda Hiroyasu , Tamura Daisuke , Aomatsu Keiichi , Kimura Hideharu , Fujita Yoshihiko , Matsumoto Kazuko , Saijo Nagahiro , Kudo Masatoshi , Nishio Kazuto
Published: January 2011

The epithelial mesenchymal transition (EMT) has emerged as a pivotal event in the development of the invasive and metastatic potentials of cancer progression. Sorafenib, a VEGFR inhibitor with activity against RAF kinase, is active against hepatocellular carcinoma (HCC); however, the possible involvement of sorafenib in the EMT remains unclear. Here, we examined the effect of sorafenib on the EMT. Hepatocyte growth factor (HGF) induced EMT-like morphologic changes and the upregulation of SNAI1 and N-cadherin expression. The downregulation of E-cadherin expression in HepG2 and Huh7 HCC cell lines shows that HGF mediates the EMT in HCC. The knockdown of SNAI1 using siRNA canceled the HGF-mediated morphologic changes and cadherin switching, indicating that SNAI1 is required for the HGF-mediated EMT in HCC. Interestingly, sorafenib and the MEK inhibitor U0126 markedly inhibited the HGF-induced morphologic changes, SNAI1 upregulation, and cadherin switching, whereas the PI3 kinase inhibitor wortmannin did not. Collectively, these findings indicate that sorafenib downregulates SNAI1 expression by inhibiting mitogen-activated protein kinase (MAPK) signaling, thereby inhibiting the EMT in HCC cells. In fact, a wound healing and migration assay revealed that sorafenib completely canceled the HGF-mediated cellular migration in HCC cells. In conclusion, we found that sorafenib exerts a potent inhibitory activity against the EMT by inhibiting MAPK signaling and SNAI1 expression in HCC. Our findings may provide a novel insight into the anti-EMT effect of tyrosine kinase inhibitors in cancer cells. Mol Cancer Ther; 10(1); 169–77. ©2011 AACR.
Chemopreventive and Chemotherapeutic Effects of Intravesical Silibinin against Bladder Cancer by Acting on Mitochondria
Authors: Zeng Jin , Sun Yi , Wu Kaijie , Li Lei , Zhang Gang , Yang Zenglei , Wang Zhiqiang , Zhang Dong , Xue Yan , Chen Yule , Zhu Guodong , Wang Xinyang , He Dalin
Published: January 2011

Intravesical chemotherapy is often used to prevent the recurrence of superficial bladder cancer after transurethral resection. A search for more effective and less toxic intravesical agents is urgently needed. We previously found the in vitro apoptotic effects of silibinin, a natural flavonoid, on high-risk bladder carcinoma cells. Here, we further explored the underlying mechanisms and examined the intravesical efficacy in the prevention and treatment of bladder cancer. Human bladder carcinoma cell line 5637, which has the same molecular features of high-risk superficial bladder cancer, was used as the model system in vitro and in vivo. Autochthonous rat model of bladder cancer induced by intravesical N-methyl-N-nitrosourea (MNU) was used to investigate its intravesical efficacy. Exposure of 5637 cells to silibinin resulted in growth inhibition and induction of caspase-dependent and -independent apoptosis, which was associated with disruption of mitochondrial membrane potential and selective release of cytochrome c, Omi/HtrA2, and apoptosis-inducing factor (AIF) from mitochondria. Silibinin also downregulated survivin and caused nuclear translocation of AIF. Oral silibinin suppressed the growth of 5637 xenografts, which was accompanied with the activation of caspase-3, downregulation of survivin, and increased translocation of AIF. Furthermore, intravesical silibinin effectively inhibited the carcinogenesis and progression of bladder cancer in rats initiated by MNU by reducing the incidence of superficial and invasive bladder lesions without any side effects, which was accompanied with proapoptotic effects. These findings identify the in vitro and in vivo antitumor efficacy of silibinin, and suggest silibinin as an effective and novel intravesical agent for bladder cancer. Mol Cancer Ther; 10(1); 104–16. ©2011 AACR.
Abstract A104: 3’, 4’, 5’-Trimethoxyflavonol (TMFol), a novel putative prostate cancer chemopreventive agent: In vitro and in vivo preclinical activity
Authors: Saad Shaban , Howells Lynne , Britton Robert G. , Steward William P. , Gescher Andreas , Brown Karen , Sale Stewart
Published: January 2011

Flavonoids occurring in many edible fruits and vegetables, typified by fisetin and quercetin, have been suggested to prevent malignancies, including those of the prostate. Only a handful of flavonoids have thus far been subjected to exhaustive pharmacological investigation, and it is unknown whether analogues exist with pharmacological properties superior to those of flavonoids studied to date. We identified TMFol as a potent inhibitor of growth of TRAMP C2, LNCaP, and PC-3 prostate cancer cells, with IC50 values of 1.3-5µM. For comparison, the respective IC50 values for fisetin and quercetin are 8-10 fold higher. Studies in mice hint at the possibility that TMFol may be useful as prostate cancer chemopreventive agent, because TMFol levels in prostate tissue after single oral administration of 240 mg/kg reached 18 nmol/g, and steady state levels after dietary intake (0.2%) were 5 nmol/g These concentrations are consistent with those eliciting growth inhibition in prostate cells in vitro. Mechanistic studies have shown TMFol to arrest cells in the G2M phase of the cell cycle and induce apoptosis. In addition, TMFol inhibited androgen receptor signalling. When nude mice bearing the TRAMP C2 cell xenograft were exposed to TMFol (0.2% in the diet) tumor volume at termination of the experiment was significantly reduced by 41% in comparison to control (p<0.001). Quercetin and fisetin had no effect, at equimolar doses, in the same mouse model. Compared to other flavonoids thus far tested as putative prostate cancer chemopreventive agents, TMFol seems superior in terms of growth-inhibitory potency, and in vivo efficacy, probably related to its metabolic stability. It therefore warrants further preclinical and clinical investigation.

Citation Information: Cancer Prev Res 2010;3(12 Suppl):A104.
Abstract A106: Efficacy of EGFr inhibitors gefitinib on development of OH-BBN-induced rat bladder tumors: Dose dependency and modulation of biomarkers
Authors: Lu Yian , Lubet Ronald A. , You Ming , Bode Ann , Steele Vernon E. , Townsend Reid , Grubbs Clinton J.
Published: January 2011

The preventive effects of the EGFr inhibitor Iressa on the induction of urinary bladder cancers induced by OH-BBN was determined. Treatment of rats with Iressa (10 mg/kg BW/day) beginning two weeks after the last dose of OH-BBN decreased the incidence of large palpable bladder cancer lesions by 95% at an 8-month observation period. Treatment with lower doses of Iressa (4 and 1.33 mg/kg BW/day) decreased the incidence of large tumors by 80 and 65%, respectively. This preventive efficacy was similarly observed when one examined an incidence curve; i.e., a large increase in tumor latency. At 3 1/2 months after the last OH-BBN dose, 75% of the rats had already developed microscopic cancers. Iressa treatment beginning at this time period also lowered the incidence of large tumors; implying that most of the effects of Iressa were during the latter periods of tumor progression. We also examined potential biomarkers modulated by short-term exposure to Iressa (10 mg/kg BW/day) in urinary bladder cancers. Three approaches were taken to identify biomarkers: (1) Alterations in EGFr related proteins directly associated with the efficacy of Iressa by immunohistochemistry. Levels of various biomarkers (phosphorylated EGFr, phosphorylated AKT, but not phosphorylated ERK levels were significantly decreased in the bladder tumors of treated rats. (2) Alterations in protein expression when comparing control and Iressa treated tumors. Employing two-dimensional gel electrophoresis and tandem mass spectrometry, it was found that proteins associated with the angiogenic effects of VEGF were decreased in tumors from treated animals. These included cytoskletal proteins nucleolin, annexin A1 and the MAP kinase (MAPKK). (3) Alterations in gene expression employing array gene technology. Numerous metabolic and cellular pathways were markedly altered by Iressa treatment, most particularly cell cycle related genes; i.e., there were strong inhibition of expression of CDC20 and both of the aurora kinases. Thus, we have identified biomarkers that might be relevant as pharmacodynamic or efficacy biomarkers in clinical prevention trials of urinary bladder cancer.

Citation Information: Cancer Prev Res 2010;3(12 Suppl):A106.
Abstract A21: Global DNA methylation analysis of bronchial epithelia of former smokers with COPD, with and without lung cancer
Authors: Vucic Emily A. , Wilson Ian M. , Chari Raj , Kennett Jennifer Y. , Lonergan Kim M. , Lam Stephen , Lam Wan L.
Published: January 2011

Introduction: Emerging evidence suggests that aberrant epigenetic regulation is involved in the development and progression of malignant and nonmalignant respiratory diseases, including chronic obstructive pulmonary disease (COPD) and lung cancer. Moreover, patients with COPD have an increased risk of developing lung cancer. Besides similar risk factors such as tobacco smoke exposure, little is known about the shared biology between COPD and lung cancer. Smoking causes aberrations in airway and lung parenchyma at both the genomic and epigenomic levels, resulting in global changes to gene expression. In this study, we hypothesize that alterations at the level of DNA methylation in airway epithelia of former smokers (FS) with COPD with and without non-small cell lung cancer (NSCLC) may be used to identify genes involved in the pathogenesis of these respiratory diseases, independent of the effects of active smoking.

Methods: Bronchial epithelial cells were obtained from brushings of small airways (< 2 mm diameter) during bronchoscopy from FS with COPD (n=22), without COPD (n=22) and patients with COPD as well as previous surgical resection of Stage I NSCLC (n=6). Illumina's Infinium Methylation (HM27) assay was used to assess DNA methylation status of 27,578 CpG sites associated with 14,475 genes.

Results: COPD patients are distinguished from non-COPD patients based on airway methylation profiles. Genes differentially methylated in airways between COPD and non-COPD patients include several modulators of aryl hydrocarbon receptor and IL6 signaling, as well as genes previously implicated in COPD, including immune chemotaxis regulators (CXCL11, CCR8) and GABA receptor signaling (GABRA5). Airway epithelial DNA from COPD patients with NSCLC compared to those without NSCLC was differentially methylated at sites encoding multiple key regulators of xenobiotic metabolism, regulators of free radical savaging/detoxification and retinol metabolic pathway components including several alcohol dehydrogenase, glutathione S transferase, and UDP glucoronosyltransferase genes.

Conclusion: Our preliminary results suggest a role for DNA methylation in the deregulation of previously identified COPD-related genes, and specifically highlight differences in airways of COPD patients with/without NSCLC corresponding to well-known smoking-related metabolomic processes. Knowledge of DNA methylation disruption will further our understanding of the etiological role of COPD in the development of lung cancer, and contribute to the development of chemo-prevention strategies targeting the biology of both COPD and NSCLC. Supported by CIHR.

Citation Information: Cancer Prev Res 2010;3(12 Suppl):A21.
Abstract A23: Allele specific regulation of esophageal cancer related gene 2 by miR-1322 in esophageal squamous cell carcinoma
Authors: Zhang Tengfei , Zhao Dan , Wang Qiming , Yu Xiying , Guo Liping , Lu Shih Hsin
Published: January 2011

Background and Aim: TCA3/TCA4 Short Tandem Repeat (STR) polymorphism in 3’ untranslated regions of esophageal cancer related gene 2 (ECRG2) was reported to be associated with esophageal squamous cell carcinoma (ESCC) incidences and the prognosis of ESCC patients. This study is to explore the mechanisms of TCA3 allele as a risk factor and poor prognosis indicator rather than TCA4 allele mainly in the functional relevant of microRNAs regulation in ESCC.

Methods: Two genotypes of luciferase reporter plasmids containing ECRG2 3’UTR (TCA3 allele and TCA4 allele respectively) were constructed and expression were compared between the two alleles in three esophageal cancer cell lines: EC9706, EC109 and KYSE150. Bioinformatics predictions of microRNAs binding were performed by software TargetScan5.1, MicroInspector, RNA22 and RNAcofold. Dual-Luciferase reporter assay with or without microRNAs mimics or inhibitors were conducted to verify the effects of predicted microRNAs on expression of different alleles. The different expression of identified microRNAs in cancer tissues and the adjacent normal esophageal tissues from 44 ESCC patients were determined by Real-Time PCR.

Results: Dual luciferase reporter assays indicated that expression level of TCA4 allele is 2-4 folds higher than TCA3 allele in all the three esophageal cancer cell lines. Bioinformatics analysis by software TargetScan5.1, MicroInspector, RNA22 and RNAcofold showed 4 potential microRNAs (miR-580, miR-1182, miR-1272 and miR-1322) binding at the TCA polymorphism site of the 3’UTR of ECRG2. Dual luciferase reporter assays indicated that miR-1182 and miR-1272 is not able to regulate either TCA3 or TCA4 allele in all three cell lines, however, miR-1322 and miR-580 dramatically down-regulated TCA3 allele expression (P <0.01) whereas slightly regulated TCA4 allele (P>0.05). The expression of miR-580 were under the detection threshold of real-time PCR analysis in most of the 44 paired tissue samples but the expression of miR-1322 were detectable, and 28 cases (63.6%) had at least 2-fold up regulation in their cancer tissues. No statistical differences were found between metastasis and non-metastasis and in different stage of disease.

Conclusions: The differential regulation of TCA3 and TCA4 allele by miR-1322 might explain the difference in cancer risk and prognosis for different genotype carriers. MiR-1322 has an important role in human ESCC by regulating tumor suppressor gene ECRG2 and miR-1322 was up regulated in esophageal cancer, which itself might severs as a tumor promoter and potential marker and therapeutic target for esophageal squamous cell carcinoma.

Citation Information: Cancer Prev Res 2010;3(12 Suppl):A23.
Abstract A29: EYA4 is a risk- and survival-associated TSG that is deleted and hypermethylated in the earliest stages of NSCLC
Authors: Enfield Katey , Wilson Ian M. , Vucic Emily A. , Chari Raj , Zhang Yu-An , You Ming , MacAulay Calum , Gazdar Adi F. , Lam Stephen , Lam Wan L.
Published: January 2011

Background: Previous familial linkage studies have identified a tumor suppressor locus at 6q23-25 in lung cancer (LC). However, no single gene has yet been implicated within this 30 Mb region. The identification of two-hit targets — genes disrupted by two mechanisms at the DNA level, such as copy number loss and hypermethylation — involved in LC risk and development will lead to better methods for risk assessment, early detection and treatment.

Methods: EYA4 was identified as a two-hit target using multidimensional genome-wide analysis combining gene dosage, DNA methylation, and gene expression data in non-small cell lung cancer (NSCLC) and early pre-malignant lesions with matched adjacent non-malignant tissue. The association of allelic variants with LC risk was assessed in data collected by the Genetic Epidemiology of Lung Cancer Consortium (GELCC), and the association of gene expression with prognosis was investigated in public data using statistical analyses. Bronchial epithelial cells of LC patients and high risk individuals were examined for early hypermethylation of EYA4. The role of EYA4 in apoptosis, and the association of EYA4 with the DNA repair genes GADD45a and γ-H2AX was assayed using stable EYA4 mRNA knock-downs in lymphoblast cell lines. Cells were either serum-starved for 12 and 24 hours followed by Annexin5/propidium iodide staining and GADD45a qRT-PCR, or irradiated, and levels of γ-H2AX protein were measured over time.

Results: Integration of NSCLC gene dosage, DNA methylation and mRNA expression showed EYA4 to be frequently affected by two-hits and significantly down-regulated. Numerous EYA4 variants were associated with familial LC, and low EYA4 expression was significantly associated with poor prognosis in multiple independent data sets. Congruent with EYA family member function, in vitro assays revealed that EYA4 knock-down cells displayed a decrease in the number of apoptotic cells following serum starvation. Further investigations led to the discovery of EYA4 hypermethylation not only in the bronchial epithelial brushings of NSCLC patients, but also in the brushings of individuals at a high risk of LC development. EYA4 knock-down led to attenuation of the GADD45a DNA damage repair response, as well as increased levels of γ-H2AX following DNA damage repair.

Conclusions: EYA4 is a gene disrupted early and frequently that maps to a locus previously associated with cancer risk. It is implicated in somatic as well as familial cancers, and is likely a tumor suppressor gene with apoptotic and DNA repair functions. The direct association of EYA4 with risk and survival as well as its early disruption underscores its relevance on a clinical level.

Citation Information: Cancer Prev Res 2010;3(12 Suppl):A29.
Abstract A42: Doses of selective estrogen receptor modulations (SERMS) required for prevention and therapy of chemically induced mammary cancers
Authors: Heckman-Stoddard Brandy , Grubbs Clinton , Johnson Karen A. , Lubet Ronald
Published: January 2011

Selective estrogen receptor modulators (SERMs) interfere with the activation of the estrogen receptor. SERMs have been shown to be effective for both treatment and prevention of breast cancer. Here we examine the ability of tamoxifen and arzoxifene, a more easily absorbed form of raloxifene, to prevent mammary tumors at a significantly lower dosage than those used for treatment. For prevention studies, female Sprague-Dawley rats (50 days of age) were injected with a single i.v. dose of MNU resulting in the development of multiple ER-positive mammary tumors beginning 7 weeks after carcinogen treatment. Rats were administered the preventive agent beginning five days after carcinogen administration. When the agents were tested at does varying between 0.15-3.3 ppm in the diet, both agents caused a dose dependent increase in preventive activity. The highest doses of tamoxifen (3.3 ppm) and arzoxifene (3.0 ppm) both decreased tumor multiplicity greater than 90%. Both of these doses are less than one-fifth the human equivalent dose (HED) based on standard FDA scaling factors. Both doses also resulted in substantial decreases in mammary gland development. These dosages were then used in a therapy setting to compare the efficacy. For these studies, MNU treated rats were allowed to develop their first palpable mammary tumor (70-150 mm2) before initiating treatment. Tumor growth or regression was followed by caliper measurements. Neither of the preventive doses of these SERMs exhibited therapeutic activity although substantially higher doses of tamoxifen (33 or 100 ppm) were relatively effective. These results are in contrast to our prior findings with other classes of agents (RXR agonists, EGFR inhibitors, and aromatase inhibitors) in which highly effective preventive doses were as effective as therapeutic doses in this model. These results demonstrate the unique dose differential regarding prevention and therapy with SERMs. Microarray analysis of tumors treated for 5 days with tamoxifen at doses of 3.3ppm or 100ppm reveals overlapping significant gene changes of similar magnitude in comparison to control treated tumors although there were significant gene changes seen at the therapeutic dose which were not observed at the lower dose. These studies raise two questions: 1) whether this class of agents might be used at significantly lower doses in a pure prevention setting as contrasted with a therapeutic setting where they were initially defined; 2) are there gene expression changes which might be used to help identify this differential dosing for prevention vs. therapy.

Citation Information: Cancer Prev Res 2010;3(12 Suppl):A42.
Abstract A50: Plantago major inhibits azoxymethane-induced aberrant crypt foci in C57BL/6 mice and modulates apoptosis in mice and human colon cancer cells
Authors: Han Peter S. , Frey Robert J. , Nishino Yuki , Kang Jonathan H. , Yeo Anthony H. , Vassantachart Janna M. , Kim Han Y. , Hausted Randy P. , Wong Brian Y. Y.
Published: January 2011

Plantago major (PM), commonly known as common plantain, is a temperate perennial herb. Traditionally, its seeds and leaves have been ingested or applied externally to treat inflammation and infection. Studies showed PM possesses inhibitory effects towards a variety of tumors and cancer cell lines, including mice Ehrlich ascites carcinoma and breast adenocarcinoma, gastric, leukaemia, ovary, melanoma, and renal human cell lines. While PM has been shown to exhibit selective cytotoxic activity against cancer cells, its quantitative potential as a chemopreventive agent on colon cancer has yet to be elucidated. In this study, C57BL/6 mice (N = 30) of 9 wk old were randomized into three groups (N = 10). Positive and experimental mice groups were injected with Azoxymethane (AOM; 5 mg/kg body weight) at the onset and second wk of the 8-wk duration of the in vivo study while the negative group received no injection. The mice in the experimental group were given daily oral feedings of 20 mg/ml of PM from the onset of the study while the negative and positive control groups were given a sterilized water placebo. All groups were sacrificed after 8 wks and 25 mm cuts of the proximal colon were analyzed for aberrant crypt foci (ACF). PM feeding significantly reduced the number of AOM-induced ACF in the proximal colon compared to the water-fed positive control AOM-induced group (7.02 ± 2.61 < 32.1 ± 9.86; P < 0.0003). The negative control, water placebo group had minimal ACF (2.0 ± 1.56) compared to the experimental and positive control groups. The potency index of the herb was calculated as 4.57. Percent inhibition of AOM-induced ACF formation of 78.1% was obtained with the PM treatment. On average, all three groups gained weight (calculated as % increase) through the course of the experiment (experimental, 6.93 < negative control 15.6 < positive control 39.4). Histological (H&E) slides of the proximal colon displayed a significantly higher apoptotic index than the positive control (AOM only) (2.0 > 0.6) and a lower mitotic index (MI) than control (1.5 < 2.8). Caspase 3 induction was identified in human colon cancer cells (COLO-205) treated with PM (5mg/ml) in chamber slides, using a specific carboxyfluorecein (FAM) labeled peptide fluoromethyl ketone (FMK) caspase inhibitor (FAM-Peptide-FMK). Caspase 3 activation was also observed in immunofluorescent slides of PM treated proximal colon tissues. Through inhibition of AOM-induced ACF via apoptosis, our data suggests PM to be a prospective chemopreventive and potential therapeutic agent for colon cancer.

Citation Information: Cancer Prev Res 2010;3(12 Suppl):A50.
Abstract A54: Orthosiphon stamineus leaf extract can prevent the development of colorectal and breast cancer in vivo
Authors: Ahmad Khadeer , Ismail Zhari , Aisheh Abdelrahim A. , Shah Amin Malik , Majid Abdul
Published: January 2011

Java tea also scientifically known as Orthosiphon stamineus (O.S) is used widely in the Southeast Asian region to treat variety of illness including kidney stones and rheumatism. Recently leaf extract of the plant species has been shown to have strong antioxidant and antiangiogenic activity causing significant inhibition in VEGF expression. Two xenograft tumor models transplanted with human breast cancer cells (MCF7) and colon cancer cells (HTC116) were administered orally 200mg/kg solvent extract of O.S. Both groups failed to show any sign of tumor growth compared to the control where almost all transplanted neoplastic tissues developed into full grown tumor. This suggests that O.S can be useful to prevent the onset of neoplasm.

Citation Information: Cancer Prev Res 2010;3(12 Suppl):A54.
Abstract A6: Nicotine stimulates lung and pancreatic cancers xenografts by systemic increase in stress neurotransmitters and suppression of the inhibitory neurotransmitter γ-aminobutyric acid
Authors: Al-Wadei Hussein A. , Al-Wadei Mohammed H. , Schuller Hildegard M.
Published: January 2011

Human lung adenocarcinoma (AC) and pancreatic ductal adenocarcinoma (PDAC) are among the most common types of human cancer and have a poor prognosis in all populations investigated. Smoking is a risk factor for both malignancies. Nicotine addiction is characterized by changes of nicotinic acetylcholine receptors (nAChRs) in the brain to increase the production of excitatory neurotransmitters while reducing inhibitory neurotransmitters. The current study has tested the hypothesis that chronic nicotine stimulates the growth of PAC and PDAC xenografts in nude mice via similar mechanisms. Groups of nude mice carrying xenografts of AC cell line NCI-H322 or PDAC cell line Panc-1 remained untreated, were given nicotine in the drinking water for 30 days in the presence and absence of daily injections with GABA or were treated with GABA alone. The diameters of xenografts were measured. Levels of noradrenaline and adrenaline, the stress hormone cortisol, the neurotransmitter γ-aminobutyric acid (GABA) in serum and xenograft tissues and levels of cAMP in the cellular fraction of blood and in xenograft tissue were assessed by immunoassays. The levels of GAD65, GAD67, nAChRs and signaling proteins downstream of β-ARs in the tumor tissue were determined by Western blotting. Our data show a significant stimulation of xenografts from both cell lines by chronic nicotine that was reversed by treatment with GABA.

Nicotine also significantly increased the systemic levels of adrenaline, noradrenaline and cAMP while decreasing GABA. The protein levels of nicotinic acetylcholine receptors α4 and α7, p-CREB and p-ERK1/2 in the tumor tissue was significantly increased by nicotine and reversed by GABA. Nicotine additionally reduced the protein levels of both GAD isozymes in tumor tissue.

In summary, our findings suggest that nicotine-induced predominance of stimulatory noradrenaline and deficiency in inhibitory GABA similar to nicotine-addiction in the brain may contribute to the development of lung adenocarcinoma in smokers and that GABA may be useful for marker-guided prevention and adjuvant therapy for PAC and PDAC.

Supported by R01 CA042829 and R01 CA130888 with National Institutes of Health.

Citation Information: Cancer Prev Res 2010;3(12 Suppl):A6.
Abstract A78: Modifiers of serum vitamin D and prostate cancer risk in African Americans
Authors: Williams Erica H. , Agurs-Collins Tanya , Dodd Kevin W. , Kittles Rick A.
Published: January 2011

Background/Objective: Prostate cancer (PCa) is the most commonly diagnosed cancer among males in the United States. Compared to European American males, African American (AA) males are at increased risk for PCa and tend to have a more aggressive form of the disease. Recent laboratory studies have suggested deficiencies in vitamin D to be related with PCa risk. Serum levels of 25-hydroxyvitamin D [25(OH)D] are considered the best indicators of an individual's overall vitamin D status. However, vitamin D metabolism and physiology are modified by genetic and environmental factors such as ultraviolet radiation (UVR), skin color, diet, body mass index (BMI) and genes involved in vitamin D synthesis. The goal of this project is to study modifiers of serum 25(OH)D levels in order to improve our understanding of the role vitamin D plays on prostate cancer susceptibility in African Americans.

Methods: This case-control study consisted of AA men (N=250) accrued from a university hospital in Washington, DC. Serum levels of 25(OH)D and prostate specific antigen (PSA) levels were assessed. BMI was calculated as weight/height2 (kg/m2). Study participants completed UVR exposure and food frequency questionnaires. Skin color was measured using a reflectance meter. Logistic regression models were used to test for associations between 25(OH)D levels and PCa risk, adjusting for ancestry, age, skin pigmentation, dietary vitamin D, supplemental vitamin D, sun exposure, and other lifestyle factors. In addition, we used linear regression models to explore determinants of serum 25(OH)D levels, including dietary vitamin D intake, ancestry, skin pigmentation and UVR exposure.

Results: There were no significant baseline differences between cases and controls for serum 25(OH)D levels, dietary vitamin D and daily servings of dairy products. Baseline differences were found for age (p<.001), alcohol intake (p<.001) and supplemental vitamin D (p=.004). Multiple logistic regression analysis found no association with serum 25(OH)D levels and PCa risk. Preliminary analysis revealed that supplemental vitamin D (p=.004) and UVR exposure (p=0.03) were determinants of serum 25(OH)D level. Also, there were potential interactions between skin color, dietary vitamin D, serum 25(OH)D levels and risk for PCa.

Conclusion: Supplemental vitamin D and UVR exposure were modifiers of serum 25(OH)D levels. However, we did not observe any significant difference in serum 25(OH)D levels between AA PCa cases and controls. This is likely due to factors such as diet, skin color, BMI and UVR exposure which strongly could influence vitamin D levels and function. Understanding the role of vitamin D in PCa risk may help to explain the higher incidence of PCa among AA males.

Citation Information: Cancer Prev Res 2010;3(12 Suppl):A78.
Abstract A8: Vasodilator-stimulated phosphoprotein Ser239 regulates matrix metalloproteinase-9 in colon cancer
Authors: Zuzga David S. , Hoffman Leah , Pelta-Heller Josh , Pitari Giovanni M.
Published: January 2011

Cancer invasion and spreading is promoted by membrane protrusion formation driven by actin polymerization and filament (F)-actin elongation, a process controlled by the actin-binding protein vasodilator-stimulated phosphoprotein (VASP). Tumor epithelial cell matrix metalloproteinase-9 (MMP-9) is a key promoter of carcinogenesis and invasion in colon cancer. Interestingly, both VASP and MMP-9 activities are regulated by intracellular cGMP signaling in colon cancer cells. However, direct functional relationships between VASP and MMP-9 are unrecognized. Here, human colon carcinoma cells were examined by immunoblotting, sucrose-gradient fractionation, confocal microscopy and imaging analysis. MMP-9 localizes at membrane protrusions and lipid rafts, cell surface microdomains specialized in signal transduction, in a cGMP-dependent manner. Activation of cGMP signaling disrupted membrane protrusion dynamics and induced intracellular MMP-9 redistribution. Strikingly, cancer cells expressing the serine-to-alanine VASP phosphomutant at Ser239, resistant to cGMP-dependent VASP phosphorylation, were insensitive to cGMP effects on membrane protrusions and MMP-9. Importantly, cGMP signaling through VASP Ser239, but not Ser157, critically regulates the ability of cancer cells to release MMP-9 in the extracellular environment. Together, these observations indicate tumor epithelial cell MMP-9, a key promoter of cancer invasion, is regulated by VASP Ser239 phosphorylation, a previously unappreciated paradigm in colon cancer biology.

Citation Information: Cancer Prev Res 2010;3(12 Suppl):A8.
Abstract B10: Side effects associated with aromatase inhibitor treatment among breast cancer patients
Authors: Gallicchio Lisa M. , MacDonald Ryan , Wood Bethany , Rushovich Errol , Helzlsouer Kathy
Published: January 2011

Introduction: Aromatase inhibitors (AIs), a first-line hormonal treatment for estrogen-receptor positive breast cancer, have clear benefit in reducing recurrence risk and increasing survival. Musculoskeletal symptoms are a well-known side effect of treatment, but the spectrum of other side effects is less well characterized. Side effects of AIs may impact quality of life and potentially medical adherence. The purpose of this study was to examine whether AI treatment was associated with side effects other than musculoskeletal symptoms.

Methods: A cohort study of 100 breast cancer patients initiating AI treatment (“cases”) and 200 women without a history of cancer (“controls”) was conducted. Questionnaire data on symptoms were collected prior to AI treatment for the cases and at the start of the study for the controls (baseline). All participants were followed for six months, with questionnaires being administered at three months and at the end of follow-up. Data on the frequency at which a number of symptoms were experienced was collected. The prevalence of each symptom at baseline among all cases and in the subgroup of cases who had not received chemotherapy was compared to controls. In addition, new onset of each specific symptom during the six month follow-up among cases compared to controls was examined. Analyses were conducted using logistic regression, and results are reported as age-adjusted odds ratios (OR) comparing cases to controls.

Results: At baseline, cases were more likely than the controls to have experienced hot flashes, decreased appetite, fatigue, fever, breast sensitivity, swelling of hands and feet, difficulty concentrating, and tendency to take naps. These differences remained when limiting the sample of cases to those who had not received chemotherapy. The initiation of AI treatment was significantly associated the new onset of the following symptoms during the follow-up period: hot flashes (OR 5.13; p<0.01), night sweats (OR 4.15; p<0.01), cold sweats (OR 4.73; p<0.01), hair loss (OR 4.74; p<0.01), leg cramps (OR 3.17; p<0.01), weight gain (OR 3.19; p<0.01), forgetfulness (OR 2.72; p=0.02), chest pain (OR 5.00; p=0.02), intestinal gas (OR 2.07; p = 0.04), cough (OR 2.58; p=0.01), depression (OR 2.67; p=0.01), breast sensitivity (OR 5.71; p<0.01), sleep disturbance (OR 2.70; p=0.01), interrupted sleep (OR 2.22; p=0.03), irritability (OR 2.39; p=0.04), and a tendency to take naps (OR 2.76; p=0.02).

Conclusions: AI treatment results in an extreme estrogen-deficient state and is associated with the onset of a number of symptoms that occur much more frequently compared to postmenopausal women of similar age without a history of cancer. These symptoms may impact adherence. This study was funded by Susan G. Komen for the Cure, AstraZeneca, and the National Cancer Institute (CA132147).

Citation Information: Cancer Prev Res 2010;3(12 Suppl):B10.