bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1408: Activation status and function of the canonical Wnt pathway in murine mammary tumor cells reexpressing Glypican-3 (GPC3)
Authors: Huvelle María A. Lago , Romero Silvina A. , Castillo Lilian , de Kier Joffé Elisa Bal , Peters María G.
Published: August 2012

GPC3 is a proteoglycan downregulated in breast cancer cells. We previously demonstrated that GPC3 reexpression inhibits in vivo metastatic potential of LM3 murine mammary adenocarcinoma cells. This event was associated in vitro with a lower motility and an increased aggregation, as well as with an enhanced senescence and apoptosis. We have previous evidence indicating that the canonical Wnt pathway is inhibited in LM3-GPC3 cells. The aim of this work was to validate the inhibition of canonical Wnt signaling in GPC3 reexpressing cells and to investigate whether differences in cell behavior are due to the regulation of GPC3 on this pathway. By means of western blots (WB) we established that LM3-GPC3 cells have 5-10 folds less phosphorylated GSK3B. Using cytoplasmic protein extracts and WB we also determined that GPC3 reexpressing cells present lower levels of cytoplasmic B-Catenin. This was further studied by immunofluorescence (IF), where it was found that while LM3-vector cells show positive signal for B-catenin mainly in nucleus and cytoplasm, this staining is restricted to the membrane and cytoplasm in LM3-GPC3 cells. In addition, a gene reporter assay employing OT/OF plasmids confirmed the canonical Wnt pathway inhibition induced by GPC3. To determine whether cell properties modulated by GPC3 are mediated by the canonical Wnt pathway, we treated cells with its activator LiCl (or NaCl as control). LiCl treatment increases 10-fold the phosphorylation of GSK3B, as compared to LM3-GPC3 cells treated with NaCl, and induces an enhance of about 50% of the canonical Wnt transcriptional activity (gene reporter assay). We confirm that although LiCl partially reverted senescence induced by GPC3 reexpression (Senescent X-Gal positive cells: LM3-GPC3+NaCl= 85% vs. LM3-GPC3+LiCl= 65%, p<0.05), it had no effects on apoptosis. We established by wound healing assays that LM3-GPC3 cells +LiCl recover their ability to migrate (Coverage of the wound: LM3-GPC3+NaCl= 15% vs LM3-GPC3+LiCl= 75%, p<0.05). Finally, LiCl treatment reduced LM3-GPC3 homotypic cell aggregation, reaching similar levels to LM3-vector clones (p<0.05). In summary, we confirm by a new methodological approach that GPC3 inhibits the canonical Wnt signaling. Our results suggest that GPC3 would be modulating senescence, migration and cell aggregation of mammary tumor cells through this pathway inhibition.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1408. doi:1538-7445.AM2012-1408
Abstract 1416: An animal model to study the effects of primary tumor excision on metastatic potential and organ-tropism of residual breast cancer cells
Authors: Zhang Yun , Jernigan Danielle , Lioni Mercedes , Garcia Fernando , Fatatis Alessandro
Published: August 2012

Because of constant improvements in early detection, the majority of breast cancer patients are diagnosed when still in a localized or regional stage and considered to be viable candidates for breast-conserving surgery or lumpectomy. As only 37% of breasts are found tumor-free following initial lumpectomy, between 22 and 59% of patients currently need re-intervention because positive or close margins are detected. In addition, re-excision or adjuvant therapies, such as local irradiation, are normally started several weeks or even months after primary surgery. During this period, the stroma at the site of tumor removal, which is characterized by altered angiogenesis, immune cells infiltration and activation of cancer-associated fibroblasts, may be selecting for cancer stem phenotypes, increasing metastatic potential and promoting the dissemination to specific secondary organs. This study aims to: 1) establish whether surgery on primary breast tumors leads to the emergence of cellular phenotypes with higher malignancy, associated with an increase in local growth, invasiveness and metastatic potential; 2) determine whether breast surgery alters the organ-tropism of residual cancer cells. For our experiments, BALB/c mice were inoculated in the mammary fat pad with 4T1 murine breast cancer cells and developed tumors that were either allowed to progress or surgically excised. Approximately 50% of animals in the surgery group presented with local recurrence, although all the excised tumors had positive margins. Local recurrences showed higher Ki-67 staining compared to their respective primary tumors and these data were in agreement with human samples of primary and recurrent breast tumors. Interestingly, the overall survival of mice in the control and surgery with recurrence groups were similar. Furthermore, macroscopic lung metastases were detected in the control and local recurrence groups, whereas mice without post-surgery recurrence failed to develop lung metastatic lesions. Finally, differences in the dissemination and growth of 4T1 cells to brain and bone between control, surgery plus recurrence and surgery without recurrence groups were investigated.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1416. doi:1538-7445.AM2012-1416
Abstract 1587: AMPK-mTOR axis as key target for chemoprevention of UV-induced skin cancer by the bioflavonoid apigenin
Authors: Tong Xin , Bridgeman Bryan B. , Smith Kimberly A. , Avram Michael J. , Pelling Jill C.
Published: August 2012

Over two million new cases of UV-induced non-melanoma skin cancer are diagnosed yearly in the USA, making it the most common form of cancer in this country. Apigenin, a nonmutagenic, naturally occurring flavonoid found in a variety of fruits and leafy vegetables, inhibits UV-induced skin cancer in mice. Our laboratory and others have shown that apigenin treatment of cells results in a wide variety of antitumorigenic and chemopreventive actions, including suppression of COX-2 expression, enhancement of p53 expression, and induction of cell cycle arrest and apoptosis. However, the primary target(s) responsible for apigenin's chemopreventive function remains elusive. For the first time, we have demonstrated that apigenin activated AMP-activated protein kinase (AMPK) in keratinocytes (mouse and human keratinocyte cell lines, primary normal human epidermal keratinocytes (NHEKs) and mouse epidermis in vivo). The activation of AMPK resulted in inhibition of mTOR signaling, which relieved the block to the autophagy pathway, further inducing autophagy in human keratinocytes. At early stages of tumorigenesis, autophagy acts as a tumor suppressor by removing damaged organelles, proteins, reducing chromosome instability, and inducing type II programmed cell death. Therefore, pharmacological induction of autophagy provides a new strategy for cancer chemoprevention. We have further demonstrated that UV radiation also induced moderate AMPK activation, suggesting that there is a protective cellular response after UV radiation alone, providing limited protection through modest AMPK activation. More intriguingly, pretreatment of keratinocytes or skin with apigenin prior to UV radiation induced AMPK activation dramatically (both in vitro and in vivo). In addition, UV markedly increased mTOR phosphorylation in SKH-1 mice epidermis, and apigenin completely inhibited UV-mediated mTOR activation. Taken together, our results demonstrate that the AMPK/mTOR axis is involved in chemoprevention of UV-induced skin cancer by apigenin, and mechanistically the activation of AMPK further induces autophagy to suppress UV-induced tumorigenesis. Identifying AMPK-mTOR axis as a key target of apigenin and targeting the autophagy pathway for cancer chemoprevention would provide a new target and strategy for the development of better treatment and prevention of skin cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1587. doi:1538-7445.AM2012-1587
Abstract 159: Macrophage Migration Inhibitory Factor (MIF) enhances proliferation and expression of tumorigenic cytokines in a bladder cancer cell line
Authors: Choudhary Shilpa , Pruitt James R. , Sielecki Thais M. , Pilbeam Carol , Taylor John Arthur
Published: August 2012

Background: MIF is a proinflammatory cytokine with regulatory properties over tumor suppressor proteins involved in bladder cancer. We have previously reported that the absence of MIF in transgenic mice or the use of a small molecule oral inhibitor of MIF (CPSI-1306, Cytokine PharmaSciences, Inc, King of Prussia, PA) leads to decreased angiogenesis and invasive potential in the BBN mouse model of bladder cancer. The objective of this study was to evaluate potential pathways of MIF activity in a cell culture model. Methods: High grade, metastatic HTB-5 bladder cancer cells were treated with rhMIF 1-100 ng/ml (CPSI). Measurements included cell counts, proliferation by 3H-thymidine incorporation (TdR), phosphorylated and total ERK by Western analysis and mRNA expression of TNF-α, IL-6, VEGF and CD74 (putative receptor for MIF) by real time PCR. CPSI-1306 (0.5-500 nM) was tested for its ability to decrease ERK phosphorylation. Results: MIF 100 ng/ml increased cell counts 2-fold (p<0.01) on culture days 2-4. MIF increased TdR, normalized to cell numbers, 30% (p<0.01). MIF (1-100 ng/ml) stimulated ERK phosphorylation in a dose dependent manner, which was abrogated by the addition of CPSI-1306 at doses above 5 nM. A specific inhibitor of ERK (PD98059, 50 μm) blocked the MIF stimulated increase in cell number. MIF treatment for 24 hrs increased mRNA expression of the agiogenic factor VEGF and the cytokine TNF-α 2-fold (p<0.01). IL-6 mRNA was unchanged. mRNA levels for the putative MIF receptor, CD74, increased 1.3 fold with treatment (p=0.01). Conclusion: MIF increased ERK phosphorylation, which was blocked by CPSI-1306, a small molecule inhibitor of MIF. MIF also increased cell proliferation via an ERK pathway. These data, along with the increased cytokine expression, provide a potential explanation for the decreased angiogenesis and invasion seen in our in vivo murine models using global knockout of MIF or CPSI-1306. This study is the first to begin to elucidate mechanistic pathways involved in MIFs role in bladder cancer and possible pathways involved in CPSI-1306 inhibition. Funding Source: ACS MRSG-08-270-01-CCE, NIH RO1DK48361

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 159. doi:1538-7445.AM2012-159
Abstract 1590: Taxifolin suppresses UV-induced skin carcinogenesis by targeting EGFR and PI3-K
Authors: Oi Naomi , Chen Hanyong , Kim Myoung Ok , Bode Ann M. , Dong Zigang
Published: August 2012

Skin cancer is one of the most commonly diagnosed cancers in United States. Taxifolin (3, 3′, 4′, 5, 7-pentahydroxy-flavanone, (2R,3R)-dihydroquercetin), which is found in many citrus fruits such as grapefruits and oranges, has been studied for multiple biological effects. However, the molecular mechanisms and direct target of taxifolin in skin cancer chemoprevention are unclear. Here we show that taxifolin suppresses UV-induced skin carcinogenesis by directly targeting epidermal growth factor receptor (EGFR) and phosphoinositide 3-kinase (PI3-K). In silico computer screening results suggest that EGFR and PI3-K are potential targets for taxifolin. In vitro and ex vivo pull-down assay data revealed that taxifolin indeed directly interacts with EGFR and PI3-K at the ATP-binding pocket, and inhibited their kinase activities in vitro. UVB-induced phosphorylation of EGFR and Akt were substantially suppressed by taxifolin in mouse skin epidermal JB6 P+ cells. Taxifolin also suppressed UVB-induced phosphorylation of mitogen-activated protein kinases (MAPKs) and cyclooxygenase-2 (COX-2) expression. Importantly, topical treatment with taxifolin to the dorsal skin significantly suppressed tumor incidence, multiplicity and volume in a solar UV-induced skin carcinogenesis mouse model. Further analysis of the solar UV-exposed mouse skin showed that the taxifolin-treated group had a substantial reduction in solar UV-induced phosphorylation of EGFR and Akt. These results suggest that taxifolin exerts potent chemopreventive activity against UV-induced skin carcinogenesis by targeting EGFR and PI3-K.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1590. doi:1538-7445.AM2012-1590
Abstract 1593: Sulforaphane protects melanocytes and skin from individuals at increased risk for melanoma from the effects of UV radiation: Role of thioredoxin reductase 1.
Authors: Cassidy Pamela B. , Quackenbush John , Campbell Jennifer , Moos Philip J. , Leachman Sancy A.
Published: August 2012

Our interest in the natural product sulforaphane (SF) began with studies of patients at high risk for melanoma. Germline loss-of-function (LOF) mutations in the highly polymorphic melanocortin-1 receptor gene (MC1R), confers a 4-fold increased risk for melanoma. Much of this increase arises from the loss of MC1R-mediated upregulation of antioxidant, pigment synthesis, DNA repair and anti-apoptotic pathways that protect epidermal melanocytes from the mutagenic effects of UV radiation. We have found that SF (likely due to its antioxidant activity) protects both normal human melanocytes, and the skin of human subjects with LOF MC1R mutations, from the harmful effects of UV light. Shave biopsies were harvested from donors with either wild-type MC1R or two LOF alleles. The epidermal tissues were treated ex vivo with UV and/or SF then analyzed histologically and by qPCR. Our results showed that SF provides significant protection from UV to both melanocytes and skin. We found also that the antioxidant protein thioredoxin reductase 1 (TR1) was significantly induced by SF. In order to explore the role of TR1 in protecting melanocytes from UV-induced damage, we have created a human melanocyte cell line that expresses a micro RNA which targets TR1. These cells display increased sensitivity to the effects of UV and differential processing of the pigment synthesis protein tyrosinase. This study demonstrates the promise of SF as an agent for protection of human skin in individuals with high-risk MC1R genotypes from the effects of UV light, and highlights the role of TR1 in this process.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1593. doi:1538-7445.AM2012-1593
Abstract 1600: Chemoprevention of 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster cheek pouch carcinogenesis by topical garcinol through inhibition of 5-lipoxygenase
Authors: Zhang Xinyan , Chen Xin , Lu Ye , Shim Joong-Youn , Sang Shengmin , Sun Zheng , Chen Xiaoxin Luke
Published: August 2012

Oral cancer is a common malignancy occurring in developing countries, and remains problematic due to lack of effective strategies of prevention and treatment. Our previous studies have shown that aberrant arachidonic acid metabolism, especially the 5-lipoxygenase (5-Lox) pathway, is involved in oral carcinogenesis, and can be targeted for cancer prevention. In order to develop potent topical agents for oral cancer chemoprevention, five known 5-Lox inhibitors from dietary and synthetic sources were evaluated for their potential efficacy when topically applied on the oral epithelium. Garcinol, a polyisoprenylated benzophenone from the fruit rind of Garcinia spp., was found to be the most promising agent based on our calculation of a theoretical activity index. In a short-term study on 7,12-dimethylbenz[a]anthracene (DMBA)-treated hamster cheek pouch, topical garcinol suppressed leukotriene B4 (LTB4) biosynthesis and inhibited inflammation and cell proliferation in the oral epithelium. In a long-term carcinogenesis study, topical garcinol significantly reduced the size of visible tumors, the number of cancer lesions, cell proliferation, and LTB4 biosynthesis. Furthermore, computer modeling showed that garcinol well fit the active site of 5-Lox, and inhibited enzyme activity through possible interactions between the phenolic hydroxyl groups and the non-heme catalytic iron. These results demonstrated that topical garcinol exerts its chemopreventive effect on DMBA-induced hamster cheek pouch carcinogenesis by inhibiting 5-Lox, inflammation and cell. Chemical modifications of garcinol may produce more potent 5-Lox inhibitors for topical use in oral cancer chemoprevention.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1600. doi:1538-7445.AM2012-1600
Abstract 1610A: Chromatin modifying agents improve the anticancer activity of resveratrol in prostate cancer cells
Authors: Savva Christiana G. , Papageorgis Panos , Constantinou Andreas I.
Published: August 2012

Resveratrol (Rsv), a dietary compound from grapes gained considerable attention as a cancer chemopreventive agent in several preclinical rodent models of prostate carcinogenesis. However, the poor bioavailability of Rsv in human plasma raised reservations about the potential health benefits of dietary Rsv. The objective of the present study was to combine Rsv with other agents, that can be given at non-toxic concentrations, in order to produce strong anticancer effects at low Rsv concentrations. For this purpose, we choose the histone deacetylase inhibitor (HDAC) suberoyl-anilide hydroxamic acid (SAHA), and the demethylating agent, 5-aza-2′-deoxycytidine (5-aza) which we evaluated in PC-3 prostate cancer cells. We found that Rsv at relatively low concentrations (2.5 μM) inhibited only marginally cell growth (<15 %). Similarly, the selected concentration of SAHA (0.5 μM) or 5-aza (0.05 μM) also produced minimal inhibition of cell growth (<17 % and <15 % inhibition respectively). A substantial decrease (> 50 %) in cellular proliferation was evident when Rsv was combined with either one of the chromatin modifying agents. Concomitant analysis of cell proliferation and apoptosis revealed that pre-treatment with SAHA or 5-aza followed by Rsv administration suspended cell growth. Cell cycle arrest was also evident which was associated with p53-indepented up-regulation of p21 and accompanied by an increase in the ratio of Bax/Bcl-2. Treatment with the pan-caspase inhibitor, z-vad.fmk did not restore cell growth, indicating that the mechanism by which these compounds trigger apoptosis is independed of caspase activation. These data suggest that the growth-inhibitory effect is mediated via upregulation of p21, down regulation of key cell cycle activators, and followed by apoptotic cell death. This combined action of Rsv might be the result of transcriptional alterations of specific genes that participate in the apoptotic and survival pathways due to the chromatin remodeling effects of SAHA and 5-aza. Our results show conclusively that, in a highly invasive prostate cancer cell line, the combination of Rsv with an HDAC inhibitor or a demethylaying agent, increases Rsv's anticancer activity. These new insights, in enhancing the anticancer effect of Rsv, may be applied to new and innovative strategies in the treatment or prevention of prostate cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1610A. doi:1538-7445.AM2012-1610A
Abstract 1613: Delta-tocotrienol delays the progression of pancreatic intraepithelial neoplasia (PanIN) lesions in a conditional KrasG12D mouse model
Authors: Husain Kazim , Centeno Barbara , Perez Marta , Lee Guo , Kazim Sabiha , Chen Dung-Tsa , Sebti Said , Malafa Mokenge
Published: August 2012

Background: We have shown that natural vitamin E γ-tocotrienol is the most bioactive tocotrienols in inhibiting the cell proliferation and induction of apoptosis in pancreatic cancer cells as well as in xenograft models. Therefore in this study we evaluated the chronic 12-month feeding of γ-tocotrienol on the prevention of the progression of PanIN lesions in a conditional KrasG12D mouse model. Methods: Offspring of LSLKRASG12D x PDX-1-Cre intercrosses (double positive by genotyping) were randomized to three groups: 1) No treatment (NT), 2) Vehicle (ethanol extracted olive oil, 1.0 ml/kg x 2/ day, PO) and 3) γ-tocotrienol (200 mg/kg x 2/day, PO). The treatment was started at the age of 10 weeks and continued for 12 months. The survival of the mice in three groups was plotted by Kaplan-Meir graph. The pancreas of KRASG12D; PDX-1-Cre mice was analyzed for the presence of murine PanINs. The apoptosis, cell cycle and signaling markers were determined by Western blotting and immunostaining. Results: No significant difference in food intake and body weight gain between γ-tocotrienol and control groups was observed during 12 months feeding. γ-tocotrienol treatment significantly increased survival of mice compared to control groups (p<0.025). Mice fed γ-tocotrienol had a significant delay in the progression of mPanIN lesions. The proportion of mPanIN-3 lesion was significantly decreased (P<0.001), whereas the relative numbers of all other lesions combined were slightly increased in the γ-tocotrienol-fed animals. γ-tocotrienol feeding also inhibited the pMEK and pERK signaling and increased p27Kip1 expression in the pancreatic tumor tissues. The apoptosis markers such as caspase-3, PARP1 cleavage, and Bax expression were increased in tumor tissues of mice treated with γ-tocotrienol compared to controls. NF-κB/p65 and the NF-κB transcriptional targets Bcl-XL was down-regulated by γ-tocotrienol treatment. The ratio of the proapoptotic (Bax) and antiapoptotic (Bcl-XL) proteins was increased almost 4-folds in the pancreatic tumor tissues compared to controls. Conclusion: γ-tocotrienol delays the progression of pancreatic cancer precursor lesions in a preclinical animal model hence warrant it's clinical use for the prevention and treatment of pancreatic cancer in high-risk patients.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1613. doi:1538-7445.AM2012-1613
Abstract 1618: Synergistic growth inhibition of human hepatocellular carcinoma cells by acyclic retinoid and GW4064, a farnesoid X receptor ligand
Authors: Ohno Tomohiko , Shimizu Masahito , Kubota Masaya , Sakai Hiroyasu , Moriwaki Hisataka
Published: August 2012

Abnormalities in the expression and function of retinoid X receptor (RXR), a master regulator of the nuclear receptor superfamily, are associated with the development of hepatocellular carcinoma (HCC). Dysfunction of farnesoid X receptor (FXR), one of the nuclear receptors that forms a heterodimer with RXR, also plays a role in liver carcinogenesis. In the present study, we examined the effects of acyclic retinoid (ACR), a synthetic retinoid targeting RXRα, plus GW4064, a ligand for FXR, on the growth of human HCC cells. We found that ACR and GW4064 preferentially inhibited the growth of HLE human HCC cells in comparison with Hc normal hepatocytes. The combination of 1 μM ACR plus 1 μM GW4064 synergistically inhibited the growth of HLE cells by inducing apoptosis and cell cycle arrest in the G0/G1 phase. The combined treatment with these agents acted cooperatively to inhibit the phosphorylation of RXRα, which is regarded as a critical factor for liver carcinogenesis, through inhibition of ERK and Stat3 phosphorylation. This combination also increased the expression levels of p21CIP1 and SHP mRNA, while decreasing the levels of c-myc and cyclin D1 mRNA in HLE cells. In addition, a reporter assay indicated that the FXRE promoter activity was significantly increased by treatment with ACR plus GW4064. Our results suggest that ACR and GW4064 cooperatively inhibit RXRα phosphorylation, modulate the expression of FXR-regulated genes, thus resulting in the growth inhibition of HCC cells. This combination might therefore be effective for the chemoprevention and chemotherapy of HCC.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1618. doi:1538-7445.AM2012-1618
Abstract 1641: Concordance between mutations identified by bar-coded multiplexed sequencing of BRAF, NRAS, and KIT in formalin fixed (FFPE) tissue from melanoma patients using the Ion Torrent Personal Genome Machine (PGM) and a real-time PCR assay
Authors: Watts George S. , Oshiro Marc M. , Cranmer Lee D.
Published: August 2012

The standard chemotherapy for metastatic melanoma is dacarbazine, however, progression free survival with dacarbazine is a dismal two months. Efforts to improve the treatment of melanoma have focused on specific molecular defects following the discovery that the majority of patients have a mutation in one of three members of the BRAF-MAPK signaling pathway: KIT, BRAF, and RAS. Recent clinical trials have shown promise for drugs that target the molecular defects in melanoma, including the recently approved mutant BRAF inhibitor vemurafenib. As new drugs that target specific mutations move through clinical trials, the opportunity to perform precision medicine is becoming a reality. To deliver on the promise shown by the new generation of drugs it will be necessary to detect specific mutations in patients so they can be matched with the right drug. To this end, we have used an Ion Torrent Personal Genome Machine to sequence the six most commonly mutated codons in melanoma (BRAF 600, NRAS 12 and 13, NRAS 61, KIT 576, and KIT 642) from formalin fixed paraffin embedded tissue of late stage melanoma patients. All six codons of interest were sequenced in forty-six patients and mutations identified by comparison to normal controls. The A375 cell line, containing the known mutation V600E in BRAF was used as a positive control. Samples were sequenced in a multiplexed format using bar coding of the amplicons which allowed 14 patients and two controls to be sequenced simultaneously on each chip. Results were confirmed by real time RT-PCR assay specific for the BRAF V600E mutation. We demonstrate the feasibility of using rapid low cost next-generation sequencing to provide robust detection of mutations in fixed patient samples.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1641. doi:1538-7445.AM2012-1641
Abstract 1643: MicroRNA processing genes and implications for complex traits
Authors: Gamazon Eric R. , Im Hae Kyung , LaCroix Bonnie , Ziliak Dana , Huang R. Stephanie
Published: August 2012

Given the fundamental role of microRNA (miRNA) expression in physiological, developmental and pathological processes, we hypothesize that genes involved in miRNA biogenesis and processing are also important in human complex traits. We identified fifty two such genes and evaluated their expression for their roles in cellular growth and sensitivity to various chemotherapeutic agents in a set of 120 International HapMap cell lines. Gene expression data were obtained through microarray-based gene expression profiling (Affymetrix GeneChip® Human Exon 1.0 ST array) and high-throughput sequencing of the transcriptome (RNA-Seq); data on sensitivity to chemotherapeutic drugs carboplatin, cisplatin, daunorubicin and etoposide were queried using a publicly available pharmacogenomics resource we developed (www.PACdb.org). We found 14 (27%) and 24 (47%) of these miRNA processing gene expression correlated with cellular growth rate and at least one of the 4 chemotherapeutic sensitivities, respectively. Regression analysis for expression between miRNA processing genes and genome-wide miRNAs (quantified using Exiqon miRCURYTM LNA arrays) suggests that the miRNA processing genes affect miRNAs and downstream target genes, leading to complex traits variability. Furthermore, from the results of genome-wide association studies we conducted between genetic variations (in the form of single nucleotide polymorphisms (SNPs) and copy number variations (CNVs)) and miRNA processing genes, we demonstrated that genetic variations that affect the expression of these miRNA processing genes as potential expression quantitative trait loci (eQTLs) also have downstream effects on human complex phenotypes, including cellular growth, sensitivity to chemotherapy and disease susceptibility.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1643. doi:1538-7445.AM2012-1643
Abstract 1655: Genome-wide association study for age at onset in breast cancer: results from the Seoul Breast Cancer Study
Authors: Sung Hyuna , Choi Ji-Yeob , Song Minkyo , Jeon Sujee , Park Sue K. , Yoo Keun-Young , Noh Dong-Young , Ahn Sei-Hyun , Kang Daehee
Published: August 2012

Introduction: While the incidence of breast cancer is relatively low in Korean women, the proportion of breast cancer that develops in younger age is much higher than in western countries. Family-based linkage study has focused on the identification of a region which is associated with the age at onset in familial breast cancer. However, genetic factors for onset age of breast cancer are still largely unknown. While genome-wide association studies (GWAS) have identified over 20 susceptibility loci for breast cancer, no previous study has examined the relationship between the common genetic variations and the age at onset in breast cancer. Methods: To identify genetic factors underlying onset age of breast cancer, we investigated the association between genetic variants and age at diagnosis in 2,155 breast cancer cases. Over 1.9 million SNPs either directly genotyped using Affymetrix 6.0 or imputed with HapMap 2.0 as a reference panel were evaluated after quality control. Tests of association were conducted with Plink v1.01 using the additive genetic model and Mach2qtl with allelic dosages in a linear regression model in which onset age was included as a dependent variable (continuous). The differences of mean age across genotype-groups were also compared using one-way analysis of variance. Results: The mean age at onset in breast cancer was 48.1 ± 9.31. The SNPs with p-value less than 1×10-5 obtained from linear regression were clustered into two regions: two SNPs (rs6684400 and rs6659875) are at 1q25.3 in intergenic region between ZNF648 and GLUL gene; the other two (rs669576 and rs667007) are at 11q25 in intronic region of NTM gene. Per-allele effect size was 1.4 ± 0.30 (p = 4.84E-06) for rs6684400 C allele and 3.1 ± 0.68 for rs669576 T allele (p = 7.11E-06). The mean age at onset for the rs6684400 G/G, C/G and C/C groups were found to be 47.2 ± 9.00, 48.1 ± 9.32 and 49.7 ± 9.59, respectively (p = 0.0001). As for the rs669576, the mean age at onset for G/G, G/T and T/T groups were 47.8 ± 9.15, 50.9 ± 10.38 and 54.3 ± 7.80, respectively (p < 0.0001) Conclusions: Our study suggests that the common genetic variants may be closely linked to age at onset in breast cancer. Further validation with a larger sample size is required to validate our result.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1655. doi:1538-7445.AM2012-1655
Abstract 1656: Genome wide identification of susceptibility loci in breast cancer survival
Authors: Jeon Sujee , Sung Hyuna , Choi Ji-Yeob , Song Minkyo , Park Sue K. , Yoo Keun-Young , Noh Dong-Young , Han Wonsik , Ahn Sei-Hyun , Kang Daehee
Published: August 2012

Background: Recently, many studies have been conducted to identify the susceptibility loci related with breast cancer risk, but few studies have examined the genetic variations for survival. Objectives & Methods: To identify the susceptibility loci for breast cancer recurrence or all-cause of death, genome wide survival analysis were performed with 2,171 histologically confirmed incident breast cancer cases. For a genetic association study, DNA was genotyped using Affymetrix SNP Array 6.0 including about 0.9 million single nucleotide polymorphisms (SNPs). We examined the association between genetic variance and disease-free survival (DFS) and overall survival (OS) using Cox proportional hazard regression model adjusting for known prognostic factors. Results: During a median of 5.8 years of follow-up, 297 women recurred and 152 women died. Three SNPs in 2 genes (ARHGAP28 and LGI4) and four intergenic SNPs were identified for DFS (Padjusted <10−6) and five SNPs in 4 genes (CADPS, KCNH7, LGI4, and TGFBRAP1) and one intergenic SNP were associated with OS (Padjusted <10−6). Most significant associated SNP with DFS was located in ARHGAP28 (Padjusted =7.40 X 10−7) and this showed decreased risk of recurrence (aHR=0.6, 95% CI=0.50-0.74). SNP of CADPS was most significantly associated with OS (Padjusted = 9.04 X 10−7), and showed increased risk of all-cause of death (aHR=2.1, 95% CI=1.56-2.83). Conclusions: Our results suggest that the promising SNPs might associate with the breast cancer recurrence and survival in Korean women. We will confirm these findings in the replication stages further.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1656. doi:1538-7445.AM2012-1656
Abstract 1669: Discovery of nasopharyngeal carcinoma susceptible variants from whole genome imputation of genome-wide association study
Authors: Su Wen-Hui , Chang Kai-Ping , Chang Yu-Sun
Published: August 2012

Nasopharyngeal carcinoma (NPC) is a multifactorial malignancy closely associated with genetic factors and Epstein-Barr virus (EBV) infection. To identify the common genetic variants linked to NPC susceptibility, we have conducted a genome-wide association study (GWAS) within the Taiwanese population, analyzing 480,365 single-nucleotide polymorphisms (SNPs). In this study, only 12 SNPs that can pass the statistical criteria for GWAS were reported and all of them located within chromosome 6 MHC region. However, those 12 SNPs can only explain a small fraction of genetic risk. The vase majority of the genetic risks remain undiscovered. Additional analytical procedures are vary likely to provide valuable information for NPC susceptibility. By utilizing 1000 genome data, we successfully expend SNP number to 37,512,779 SNPs. After removed SNPs with low quality (info <0.5) and no heterozygosity, we discovered additional 57,212 SNPs from whole genome imputation including 3,751 SNPs with P values <0.05. SNPs located in chromosome 1, 2, 3, 8, 15, 17 were selected for further validation. This work taking advantage of current genome sequencing database and significantly expand the range of associated SNPs that can also increase the chance to discover true association signals. Following functional investigation and experimental application of these findings are expected to produce new advances in the prevention and treatment of NPC.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1669. doi:1538-7445.AM2012-1669
Abstract 1675: Effects of calcium and vitamin D on TGFβ and TGFβ1 expression in rectal mucosa of sporadic colorectal adenoma patients: A randomized controlled trial
Authors: Tu Huakang , Daniel Carrie , Ahearn Thomas , Bostick Roberd , Gonzalez-Feliciano Amparo
Published: August 2012

Aim: To estimate the effects of supplemental calcium and vitamin D3 on the expression of TGFβ and TGFβ1 (growth-promoting and -inhibiting autocrine/paracrine growth factors, respectively) in the normal-appearing colorectal mucosa in sporadic colorectal adenoma patients. Methods: We conducted a pilot, randomized, double-blind, placebo-controlled, 2 x 2 factorial clinical trial (n = 92) of calcium 2 g and/or vitamin D3 800 IU/day versus placebo over 6 months. TGFβ and TGFβ1 expression was measured in biopsies of normal-appearing rectal mucosa using automated immunohistochemistry and quantitative image analysis at baseline and 6-months follow-up. Results: The mean overall expression of TGFβ1 in the whole crypts increased by 14% (P = 0.25), 19% (P = 0.17), and 22% (P = 0.09) in the calcium, vitamin D, and calcium plus vitamin D groups, respectively, relative to the placebo group. The increases in TGFβ1 appeared to be uniform along the full length of the crypts. While there were no substantial changes in the overall expression of TGFβ in the whole crypts, in the calcium, vitamin D, and calcium plus vitamin D groups, TGFβ decreased by 14% (P = 0.35), 2% (P = 0.86), and 1% (P = 0.93), respectively, in the upper 40% (differentiation zone) of the crypts, but increased by 29% (P = 0.30), 38% (P = 0.17), and 25% (P = 0.32) in the bottom 60% of the crypts (proliferative zone). Also, a TGFβ/TGFβ1 ratio was created as an indicator of the balance between the growth-promoting and -inhibiting factors; in the upper 40% of the crypts it was found to have decreased by 35% (P = 0.24), 25% (P = 0.42), and 51% (P = 0.09) in the calcium, vitamin D, and calcium plus vitamin D groups, respectively, relative to the placebo group, but not to have changed substantially in the whole or bottom 60% of the crypts. Conclusions: These preliminary results suggest that supplemental calcium and vitamin D3 may increase the expression of TGFβ1, shift the expression of TGFβ downward in the colorectal crypts, and decrease the ratio of TGFβ to TGFβ1 in the upper 40% (differentiation zone) of the crypts in the normal-appearing colorectal mucosa of sporadic colorectal adenoma patients, and support further investigation of calcium and vitamin D3 as chemopreventive agents against colorectal neoplasms.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1675. doi:1538-7445.AM2012-1675
Abstract 1680: Age-associated gene expression in normal breast tissue provide insights regarding associations between aging and aggressive tumor characteristics
Authors: D'Arcy Monica , Pirone Jason R. , Stewart Delisha A. , Casbas-Hernandez Patricia , Gould Michael N. , Jerry Joseph Jerry , Yaswen Paul , Schneider Sallie Smith , Troester Melissa A.
Published: August 2012

Age is the strongest breast cancer risk factor, with overall breast cancer risk increasing steadily beginning at approximately 30 years of age. Though several genomic studies have characterized differences in the biology of tumors arising in younger vs. older women, understanding of age-related gene expression in the non-malignant tissue is lacking. Because non-malignant tissue represents the microenvironment and/or field from which the tumor arises, an improved characterization of the age-related change in normal breast may provide suggestions of important pathways in the etiology of distinct breast cancer subtypes. To identify age-related changes in the breast microenvironment, 96 normal tissue specimens from reduction mammoplasty patients ranging in age from 14 to 70 were assayed by gene expression microarray. Significant associations between gene expression levels and age were identified for 802 genes (481 increased and 321 decreased with increasing age). Enriched functions included ‘aging of cells’, ‘shape change’, and ‘chemotaxis’, and enriched pathways included Wnt/beta-catenin signaling, Ephrin Receptor Signaling, Oncostatin M signaling, and JAK/Stat Signaling. Applying the age-associated genes to publicly available tumor datasets, the age-associated pathways defined two groups of tumors with distinct survival. The poor prognosis tumor group shared features of ‘younger’ breast tissue gene expression, providing a biological link to qualitative patterns in age at incidence. Aggressive tumors common in younger women (such as ER negative and basal-like breast cancers) harbor gene expression patterns that reflect the normal tissue from which they arise. For the young, these signatures portend more aggressive disease. These data show that studies of normal tissue gene expression can yield important insights about the pathways and biological selection factors that are relevant during tumor progression.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1680. doi:1538-7445.AM2012-1680
Abstract 169: Inhibition of focal adhesion kinase suppresses gastrin-releasing peptide receptor-mediated liver metastasis in neuroblastoma
Authors: Lee Sora , Qiao Jingbo , Paul Pritha , Chung Dai H.
Published: August 2012

We have previously reported that high expression of gastrin-releasing peptide receptors (GRP-R), a member of the G-protein coupled receptor family, is observed in more undifferentiated human neuroblastoma. We have also found that GRP-R-mediated cell signaling plays a critical role in neuroblastoma cell transformation; however, its molecular mechanisms are unknown. Here, we sought to determine the role of focal adhesion kinase (FAK), a non-receptor protein tyrosine kinase, in GRP-R-mediated neuroblastoma cellular transformation. METHODS. Using human neuroblastoma tissue sections, the correlation of GRP-R and FAK expression was examined. Human neuroblastoma cell line, BE(2)-C and SK-N-SH were used for our experiments. Using transfection with Lipofectamine 2000, the level of knockdown and overexpression of GRP-R and FAK were modulated. MTT assay was carried out to measure the cell viability of neuroblastoma cells, and soft agar colony formation assay was performed to determine the capacity of cell transformation. Transwell migration and wound healing assay were employed to study cell motility. Rescue experiments by overexpression of FAK in GRP-R silencing cells were conducted to further validate the role of FAK in neuroblastoma cell transformation. Using FAK inhibitor and GRP-R agonist, animal experiment was performed. RESULTS. FAK expression correlated with GRP-R expression in BE(2)-C and SK-N-SH human neuroblastoma cells. GRP treatment induced phosphorylation of FAK at Y397 in both cell lines. Overexpression of GRP-R in SK-N-SH cells, which are known to express low endogenous levels of GRP-R, increased the levels of phosphorylated and total FAK. Moreover, overexpression of FAK increased cell viability and colony formation of SK-N-SH cells. These effects were reversed when cells were co-transfected with FAK siRNA in BE(2)-C cells, which endogenously express higher levels of GRP-R. Conversely, silencing of GRP-R decreased the levels of phosphorylated and total FAK in BE(2)-C cells. Interestingly, in cells with GRP-R silencing, FAK overexpression significantly enhanced cell viability and colony formation. GRP-R-mediated liver metastasis suppressed by FAK inhibitor in vivo. CONCLUSIONS. Our results suggest that FAK is a critical downstream regulator of GRP-R-mediated tumorigenesis. Furthermore, GRP-R and FAK expression correlate with morphological changes implicated in the malignant transformation of human neuroblastoma cells. Importantly, inhibition of FAK can suppress GRP-R-induced tumor progression in neuroblastoma in vivo.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 169. doi:1538-7445.AM2012-169
Abstract 1708: LINE-1 methylation level is a molecular marker with little intra-patient heterogeneity in primary and synchronous metastatic colorectal cancer
Authors: Matsunoki Aika , Kawakami Kazuyuki , Kotake Masanori , Kaneko Mami , Watanabe Go , Minamoto Toshinari
Published: August 2012

Introduction: Epigenetic alterations including hypermethylation of gene promoter regions and global hypomethylation of DNA are common in colorectal cancer (CRC). We recently reported that long interspersed nuclear element 1 (LINE-1) methylation level in tumor, a surrogate of global methylaiton, predicts prognosis and response to oral fluoropyrimidines of CRC patients. This suggests that the LINE-1 methylation level is a useful marker for tailored therapy in CRC. However, the LINE-1 methylation level in tumor may be heterogeneous because the DNA methylaion is reversible, which is a problem when the LINE-1 methylation level in tumor is used for decision making in clinic. In this study we investigated LINE-1 methylation level in cancer cells isolated from multiple tumor sites to evaluate intra-patient heterogeneity of the LINE-1 methylation level in tumor tissues. Methods: Primary tumor, metastatic lymph nodes, and distant metastases in 41 CRC patients were used for measurement of LINE-1 methylation level. Cancer cells in primary tumors were collected from tumor center and invasion front by laser capture microdissection (LCMD). Cancer cells in metastatic lymph nodes and distant metastases were also collected by LCMD. The LINE-1 methylation level was measured by multicolor MethyLight assay following bisulfite modification of DNA and was correlated with clinicopathological parameters. Results: There was no significant difference in the LINE-1 methylation level between cancer cells from the center and those from invasion front of the primary tumor tissue. In the same patient, there was no significant difference in the LINE-1 methylation level between the samples from primary tumor, metastatic lymph nodes, and distant metastases. The LINE-1 methylation level was decreased as the tumor stage advances. Discussion: The results suggest that the LINE-1 methylation level is a molecular marker with little intra-patient heterogeneity in primary and synchronous metastatic colorectal cancer. Prediction of patients’ prognosis using LINE-1 methylation level may be possible by multiple tumor specimen such as biopsy specimen by colonoscopy and surgically resected tumor. Measuring LINE-1 methylation level in primary tumor may be useful for prediction of response to fluoropyrimidines in metastatic tumor.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1708. doi:1538-7445.AM2012-1708