bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3782: Chloroquine induces defective autophagic flux and enhances pancreatic cancer cell death induced by pegylated-arginine deiminase
Authors: Daylami Rouzbeh , Virudachalam Subbulakshmi , Bold Richard J.
Published: June 2012

We have previously shown that a majority of human pancreatic adenocarcinomas are deficient in argininosuccinate synthetase (ASS), a key enzyme in arginine synthesis and therefore sensitive to arginine deprivation by arginine deiminase (ADI). ASS deficient cell lines undergo cell death when deprived of arginine, but also undergo autophagy. The role of autophagy and its contribution to cell death is controversial. We hypothesized that autophagy is protective in the setting of arginine deprivation and that inhibition of autophagy by hydroxychloroquine (ChQ) would increase cell death. Methods: The human pancreatic cancer cell line MIA-PaCa2 was treated in vitro and in vivo with pegylated arginine deiminase (PEG-ADI) alone or in the presence of ChQ. Cell death was measured by propidium iodide-FACS and apoptosis was evaluated by Annexin V-PI flow cytometry. Caspase 3 cleavage was evaluated by western blotting and ELISA as was expression of LC3. In vivo experiments were conducted by creation of subcutaneous xenografts in athymic mice followed by treatment with intraperitoneal injections of PBS, PEG-ADI, ChQ and combination of PEG-ADI and ChQ. At sacrifice, tumors were removed for analysis. Tumor lysates were analyzed by western blot for caspase 3 and p62. Immunohistochemistry for activated caspase 3 and DNA fragmentation (TUNEL) were also performed. Results: In vitro treatment of MIA-PaCa2 with PEG-ADI induced caspase dependent cell death as well as autophagy at 72 hours. ChQ at low doses inhibited autophagy induced by PEG-ADI, but increased cell death. In vivo, addition of ChQ to a sub-therapeutic dose of PEG-ADI increased tumor suppression decreased autophagy and increased caspase 3 activation compared to either treatment alone. Conclusion: Arginine deprivation induces autophagy and cell death in cell lines deficient in ASS. Autophagy appears to play a protective role, and its inactivation by hydroxychloroquine leads to enhanced tumor suppression.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3782. doi:10.1158/1538-7445.AM2011-3782
Abstract 3786: LC3B-positive autophagosomes are a common feature of solid tumors and associated with proliferation, metastasis and poor outcome
Authors: Lazova Rossitza , Camp Robert , Siddiqui Summar , Klump Vincent , Pawelek John M.
Published: June 2012

Interest in autophagy and cancer has surged in recent years. Autophagy provides a source of nutrients in times of metabolic stress. A paradox for autophagy in cancer is that while autophagy is a conserved pro-survival mechanism, it can also act in tumor suppression as is the case with the autophagy-associated beclin-1 protein. However, there is growing evidence supporting a pro-survival role for autophagy in tumor progression, for example in gastrointestinal tumors and invasive melanomas wherein autophagosomes are abundant. Here we expanded previous studies on autophagosomes in malignancies to include nearly 2000 pathology specimens from 20 different cancers, analyzing for associations of autophagosomes with tumor progression. Methods. Individual pathology specimens and tumor tissue microarrays (TMAs) were stained for the autophagosome marker LC3B and the proliferation marker Ki-67 through standard IHC procedures. Automated quantitative analyses (AQUA) used fluorescence-tagged antibodies. Results: In multitumor TMAs, about 85% of all tumors showed positive staining of LC3B in a granular pattern. In melanoma TMAs, LC3B was elevated in lymph node metastases. In breast carcinoma TMAs LC3B was elevated in node-positive compared to node-negative primaries and associated with increased nuclear grade, and worse patient outcome. LC3B and Ki-67 expression correlated in both melanoma and breast carcinoma (p < 0.0001), indicating a strong association between autophagy and proliferation. Supporting this, mitotic figures were largely detected in LC3B-positive tumor cells. Conclusions. While it is clear that autophagy can play a tumor suppressor role in some settings, we posit that these results are most consistent with a pro-survival role for autophagy, at least in advanced malignancies. In cancer, autophagy is thought to be part of the pro-survival integrated stress response known as aerobic glycolysis or the Warburg effect whereby cells utilize glycolysis rather than aerobic respiration for ATP production. Since some 90% of malignancies constituitively express the Warburg effect and, as shown herein, a similar number express LC3B-positive autophagosomes, the results are most consistent with autophagy being an integral, active part of this response. That autophagosomes are expressed in proliferating tumor cells further supports this proposal. In any case, the widespread occurrence of autophagosomes in cancer shown herein, and by others, underscores the importance of autophagy as a target in the design of new anti-cancer therapies.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3786. doi:10.1158/1538-7445.AM2011-3786
Abstract 3789: Induction of autophagy increases the radiation sensitivity of head and neck cancer cells via growth arrest
Authors: Nam Hae Yun , Lee Yoon Sun , Lee Hyang Ju , Im I-Rang , Kwak Jihye , Kim Seong Who , Kim Sang Yoon
Published: June 2012

Autophagy is frequently activated in cancer cells resistant to various apoptotic stimuli such as ionizing radiation, which supports the premise of enhancing the efficiency of radiotherapy by interventional means of autophagy regulation. In this study, we investigated whether autophagy pathway mediates the radio-sensitizing effect in head and neck cancer cells, and how the mechanism underlying the enhanced sensitivity works in cancer cells.

The cellular responses following the ionizing radiation were examined by cell morphologic change, cell proliferation, clonogenic survival, cell cycle distribution, and expression level of several corresponding marker proteins in head and neck cancer HN-9 cells. Induction of autophagy was performed by down-regulating the mTOR signaling pathway using mTOR inhibitors such as rapamycin and PP242. Also, autophagy was inhibited by blocking the formation of autophagic vacuole (3-MA) or by disturbing the autophagosome maturation (bafilomycin A1, pepstatin A/E-46d). The change of autophagic flux was evidenced by the level of autophagy-related proteins and the morphological manifestation of autophagy. Upon exposure to radiation, the number of autophagic vacuoles was increased greatly in radiation dose- and time-dependent manners; however, the increase of apoptotic cells was not observed even in cells exposed to 12 Gy. In the meanwhile, the initial inhibition of cell cycle was maintained at slightly enhanced G1 phase. In some of the cases, it was restored to normal states, which implied that HN-9 cells adopted the mode of autophagy as a survival strategy against radiation. However, unexpectedly, further augmentation of autophagy pathway through the combination of either rapamycin or PP242 and radiation toward HN-9 cells hindered rather than enhanced clonogenic survival and cell proliferation, accompanying prolonged G1 arrest. Taken together, these results suggest that either induction of autophagy could serve as a therapeutic strategy to enhance radio-sensitivity, which is possibly due to irreversible cell cycle arrest following the excessive activation of autophagy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3789. doi:10.1158/1538-7445.AM2011-3789
Abstract 379: Target delivery of multistage nanovectors to tumor-associated macrophages and endothelial cells in orthotopic model of human pancreatic cancer
Authors: Yokoi Kenji , Godin Biana , Alexander Jenolyn F. , Hu Tony , Liu Xuewu , Fidler Isaiah , Ferrari Mauro
Published: June 2012

Pancreatic cancer is one of the most aggressive malignancies in humans with a 1-year survival rate of 18%, and a 5-year survival rate of less than 3%. Clearly, there is an urgent need to develop novel therapies for this disease. In patients, the tumor microenviroments consist of 70 to 90% stroma tissue supporting growth, invasion and metastasis of cancer. Therefore stroma is a potential target for therapy. The stroma consists of various cellular elements, including tumor associated macrophages and endothelial cells. Identification of molecular targets overexpressed in stroma cells holds great potential for design of specific nanocarriers to attain homing of therapeutic and imaging agents to the tumor. Physico-chemical characteristics of the nanovectors greatly affect their in vivo behavior and we have recently shown that carrier geometry plays an important role in its biodistribution.

In this study, we found increased expression of CD59 in tumor associated endothelial cells as well as infiltrating cells in the stroma as compared to uninvolved pancreas in clinical specimen of pancreatic cancer patients. We explored this dual targeting effect using L3.6pl human pancreatic cancer cells implanted into the pancreas of nude mice. To produce a chronic disease with high proportion of stroma, we injected only 5 × 104 viable cells that produced visible tumors after three months (chronic disease model). Increased stroma formation was found only in the chronic disease model, but not when 1 × 106 cells were injected (acute model). In the chronic model, immunofluorescence analysis of tumor confirmed the increased expression of Ly6C, mouse homolog of CD59, in tumor associated endothelial cells as well as in macrophages in stroma. In the next experiment, we decorated the surface of porous silicon nanovectors with Ly6C antibody. Targeted nanovectors injected intravenously accumulated to tumor associated endothelial cells expressing Ly6C 15 minute after injection. At 4 hours after administration, 9.8 ± 2.3 % of injected dose/g tumor of the Ly6C targeting nanovectors accumulated in the pancreatic tumors as opposed to 0.5 ± 1.8 % of control nanovectors with non targeting antibody. Immunofluorescence analysis of tumor revealed that Ly6C targeting nanovectors were engulfed by tumor associated macrophages which also express Ly6C in stroma.

Collectively, these results suggest that Ly6C (or CD59) expressed on tumor associated endothelial cells and macrophages can serve as a novel dual target for pancreatic cancer. These experiments are on going.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 379. doi:10.1158/1538-7445.AM2011-379
Abstract 3792: Specific amino acid restriction differentially induces autophagy in DU145 and PC3 human prostate cancer cell lines
Authors: Fu Ya-Min , Lin Huimin , Meadows Gary G.
Published: June 2012

We previously found that selective amino acid restriction differentially induces death of DU145 and PC3 human prostate cancer cells. Herein we examined the hypothesis that this differential cell death was associated with induction of autophagy. By detecting the immunofluorescence of microtubule-associated protein 1A/1B light chain 3 (LC3) by confocal microscopy, this study shows that selective amino acid restriction also differentially induces autophagy in DU145 and PC3 human prostate cancer cell lines.

In DU145 cells, restriction of tyrosine and phenylalanine (Tyr/Phe), glutamine (Gln), and methionine (Met) induces autophagy. Addition of pyruvate significantly increases Tyr/Phe restriction-induced autophagy and significantly reduces Met and Gln restriction-induced autophagy. All of the amino acid restrictions induce cell death in DU145 cells. Pyruvate, a major metabolite of glucose metabolism, significantly reduces Tyr/Phe and Gln restriction-mediated cell death, but not Met restriction-medicated cell death.

Tyr/Phe and Gln restriction induces autophagy in PC3 cells. Addition of pyruvate does not alter Tyr/Phe restriction-induced autophagy but significantly increases Met and Gln restriction-induced autophagy. Only Met restriction induces cell death in this cell line, and pyruvate addition reverses this effect.

In summary, the present study indicates 1) cell line specific differences between DU145 and PC3 cells in the induction of autophagy by specific amino acid restriction, 2) lack of association between induction of autophagy and cell death, and 3) differential effects of pyruvate on autophagy and cell death. (This study was supported by grant R01CA101035 from the National Cancer Institute).

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3792. doi:10.1158/1538-7445.AM2011-3792
Abstract 3801: Rhomboid domain containing 2 (RHBDD2) over-expression is associated to colorectal cancer progression and drug sensitivity to 5-FU treatment
Authors: Lacunza Ezequiel , Croce Maria V. , Zwenger Ariel , Segal-Eiras Amada , Abba Martín C.
Published: June 2012

Colorectal carcinoma (CRC) is the second leading cause of death among malignancies in the world. Postoperative chemotherapy is widely accepted as the standard modality for the treatment of this disease; however 50% of patients within an oncology protocol will develop tumor metastatic dissemination. The drug 5-fluorouracil (5-FU) is one of the most commonly used agents for the treatment of CRC at early and advanced tumor stages, but clinical resistance is a major limitation. The identification of novel predictive biomarkers of resistance to 5-FU treatment would allow developing new therapies and improving the quality of life for CRC patients.

The aim of this study was to evaluate the role of RHBDD2 gene expression in human colorectal carcinogenesis and its effect on treatment with chemotherapeutic agent 5-FU. Firstly, we analyzed a dataset of 430 colorectal samples (32 healthy control, 355 primary CRC and 43 metastatic CRC) derived from two independent public available gene expression studies: Bittner et al., 2005 (GEO Acc.#GSE2109), and Sabates-Bellver et al., 2007 (GEO Acc.#GSE8671). A statistical significant increase in RHBDD2 expression was detected between normal colorectal samples and CRC specimens with and without metastasis (p<0.001). In order to validate these data, we performed an immunohistochemical analysis on 140 colorectal tissue samples (5 normal tissues, 20 benign lesions and 115 colorectal carcinomas). We detected high RHBDD2 protein expression in tumor samples compared with normal and benign lesions (p<0.005). Interestingly, a subset of 33 CRC specimens which were obtained from patients treated with 5-FU prior to surgery, showed a higher intensity of reaction compared with the other CRC samples (p<0.001). To analyze this finding, RHBDD2 5-FU sensitivity was evaluated by Real Time quantitative RT-PCR in the colon carcinoma cell lines Colo205 and Caco-2. Cells were incubated with 5-FU (50 to 200 µM) for 48 and 72Hs. We detected a dramatic up-regulation of RHBDD2 mRNA levels in both cell lines after being exposed to 5-FU (p<0.001). Furthermore, we analyzed the phenotypic effects of silencing RHBDD2 gene expression with and without 5-FU treatment in both cell lines over cell proliferation and apoptosis.

In conclusion, our findings suggest that RHBDD2 over-expression might play a role in colorectal neoplastic progression, modulating the response of colon cancer cells to 5-FU treatment. Further studies are needed to evaluate the relevance of RHBDD2 gene expression as a predictive biomarker as well as a therapeutic target to enhance drug efficacy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3801. doi:10.1158/1538-7445.AM2011-3801
Abstract 3813: Metabolites as biomarkers for ovarian cancer diagnosis and treatment
Authors: Kakar Sham S. , Fong Miranda Y.
Published: June 2012

Ovarian cancer is the most lethal malignancy of the female reproductive track, mainly due to a lack of a reliable detection method during the early stages of this disease when it is most treatable. Currently, most cases of ovarian cancer are detected at stage IV due to a lack of symptoms. As current detection methods identify 50-60% of early stage ovarian cancer, it is important to identify novel biomarkers that are both specific and sensitive. In this study, we compared the comprehensive metabolic profile in normal, primary epithelial ovarian cancer (EOC), and metastatic tumors resulting from primary ovarian cancer (MOC) using gas chromatograph mass spectroscopy (GC/MS) or liquid chromatograph mass spectroscopy (LC/MS). Despite a small sample size, we were able to detect significant changes in energy utilization, shifting from oxidative phosphorylation to fatty acid utilization marked by increases in several fatty acids and carnitine metabolism_such as carnitine, acetylcarnitine, and propionylcartine _and by increases in fatty acid metabolism. EOC and MOC also displayed an enhanced oxidative stress response as indicated by increases in 2-aminobutyrate, phenylpyruvate, phenyllactate (PLA), and several isoforms of tocopherols. By using this method, we have also identified novel metabolites in the ovary, specifically N-acetylasparate (NAA) and N-acetyl-aspartyl-glutamate (NAAG), for which a biological function has yet to be determined. Using these findings, potential biomarkers can be identified for both preliminary cancer diagnosis and metastatic disease progression.

Supported by NCI CA 124630 (SSK).

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3813. doi:10.1158/1538-7445.AM2011-3813
Abstract 3816: Genetic variants within JAZF1 are associated with differential binding of androgen receptor, altered mRNA expression and risk of prostate cancer
Authors: Tarway McAnthony D. , Tang Wei , Fu Yi-Ping , Liu Luyang , Porter-Gill Patricia , Hall Jennifer , Ambs Stefan , Prokunina-Olsson Ludmila
Published: June 2012

Genome-wide association studies (GWAS) identified single nucleotide polymorphism (SNP) rs10486567 within the JAZF1 gene on chromosome 7p15 as being associated with increased risk of prostate cancer in individuals of European ancestry (p=2.14×10-6, Thomas, 2008; p=7.79×10-11, Prokunina-Olsson, 2010). We now examined two additional SNPs, rs7808935 and rs67152137 that are in strong linkage disequilibrium with rs10486567 (r2∼1.0) and located in a 2 Kb non-coding genomic region of intron 2 of JAZF1. By chromatin immunoprecipitation (ChIP) assays in LNCaP and DU-145 prostate cancer cell lines, we established that all three SNPs regions bind androgen receptor, but only rs7808935 increased allele-specific binding, with the risk T allele enhancing binding (1.4-fold, p=0.021). We cloned the 2Kb genomic region encoding various haplotypes of the three SNPs into a luceferase reporter assay, and discovered that the risk haplotype increased the reporter activity by 2-fold (p=6.98×10-5). mRNA expression of JAZF1 in normal prostate tissue samples was increased in carriers of the risk haplotype (1.51-fold, p=0.015, n=59). The three associated SNPs are located in large intron 2 and about 250 Kb away from JAZF1 promoter region. To further explore the molecular relationships between the three prostate-cancer associated SNPs, mRNA expression of JAZF1 and risk of prostate cancer, we are currently performing analysis of long-distance chromatin interactions that involves the associated JAZF1genomic region.

In conclusion, we hypothesize that the prostate-cancer associated variants of JAZF1 influence androgen receptor transcriptional activity and its effect on JAZF1. Our current research should identify mechanisms of how this affects JAZF1 mRNA expression and carcinogenesis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3816. doi:10.1158/1538-7445.AM2011-3816
Abstract 3824: Molecular cytogenetic study of a new human rhabdomyosarcoma cell line, HS-RMS-2, with various highly- amplified DNA sequences
Authors: Taguchi Takahiro , Sonobe Hiroshi , Takaoka Eiji , Akimaru Kunihiro , Sakamoto Shuji , Shuin Taro , Daibata Masanori , Tominaga Akira
Published: June 2012

Molecular cytogenetic characterization was carried out on a rare pleomorphic type of the human rhabdomyosarcoma cell line, HS-RMS-2, which had been newly established in our laboratory. Conventional G-banding and multicolor-FISH (M-FISH) analyses showed that cells had a complicated chromosomal constitution. Comparative genomic hybridization (CGH) revealed seven highly amplified regions, which suggested a simultaneous amplification of several oncogenes in the same cell. The locations of highly amplified sequences were as follows; 1p36.1-36.2, 1p31-32, 1q21-31, 8q12-21, 8q24-qter, 11q12-13, 12q13-14, and 18q12-22. Reverse FISH painting, using the probe which was regenerated from long marker chromosomes by chromosome microdissection, showed that original sites of four of seven possible genes, which derived from highly amplified regions of chromosomes, located on chromosomes, 1p31-32, 11q13, 12q13 and 18q21. FISH using BAC and cosmid probes (int-2, c-jun and c-myc) which located on 11q12-13, 1p31-32 and 8q24, respectively, demonstrated that those oncogene DNA sequences were clearly amplified. By PCR, amplification of oncogenes of BCL1, BCL2, CHOP, hst-1, int-2, c-jun and c-myc were seened. Also RT-PCR analysis for gene expression showed that the genes located at 1p31-32 and 8q24 were overexpressed. These seven amplified genes may play a important role in the pathogenesis of this rare pleomorphic type of rhabdomyosarcoma.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3824. doi:10.1158/1538-7445.AM2011-3824
Abstract 3826: Brevican absence and YKL-40 over-expression are associated to the mesenchymal profile and in vitro 3D-neurosphere growth of human glioma
Authors: Martinez Paloma Fernandez , Casans Cristina Zahonero , de la Rosa Natividad Pozo , Ayuso Angel , de Sola Rafael Garcia , Sanchez Juan Manuel Sepulveda , Gomez Pilar Sanchez
Published: June 2012

DNA microarray-based molecular profiling have recently uncovered two well-differentiated subsets of human glioma having distinct clinical outcome: those displaying neuronal lineage markers and having longer survival (proneural), and those enriched for neural stem cell markers and having short survival (mesenchymal). Genes allowing the distinction of both glioma subsets were herein used to further explore their clinical and biological significance with regard to survival and tumorigenic potential through 3D-neurosphere in vitro assay. Surgical samples from 72 patients with glioma (tumor tissue and tumor-unaffected brain tissue from the same patients) were used. The expression level of most relevant genes, including cancer stem cell markers, matrix and tumor progression genes were analyzed by quantitative RT-PCR. Brevican (BCAN), Delta-like 3 (DLL3) and oligodendrocyte lineage transcription factor 2 (Olig2) genes were associated to the proneural profile (Spearman correlation between them indicated a robust P-value: DLL3-BCAN: 2.17E-10; Olig2-BCAN: 6.94E-9; Olig2-DLL3: 5.03E-11). In contrast, Fibronectin 1 (FN1) and cartilage glycoprotein-39 (YKL-40) gene over-expression defined the mesenchymal profile in an effective way (Spearman correlation YKL-40-FN1 p-value: 2.65E-4). Survival increased by two-fold in patients having BCAN gene over-expression. Moreover, BCAN gene expression prevented in vitro growth of glioma cells as 3D-neurospheres. These data demonstrate a correlation between BCAN, DLL3 and Olig-2 gene expression and the proneural glioma profile, while absence of BCAN expression and YKL40 overexpression was associated to the mesenchymal profile and in vitro neurosphere growth of human glioma.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3826. doi:10.1158/1538-7445.AM2011-3826
Abstract 3830: Type 2 cGMP-dependent protein kinase regulates proliferation in the colon
Authors: Wang Rui , Kwon In-Kiu , Browning Darren D.
Published: June 2012

Signaling through cGMP has previously been found to play a role in tissue homeostasis in the intestinal mucosa but the mechanism is not known. Type 2 cGMP-dependent protein kinase (PKG2) is a major cGMP effector in the intestinal epithelium but its role in tissue homeostasis has not previously been examined. The present studies have tested the importance of PKG2 in the regulation of proliferation and differentiation in the mouse colon and in colon cancer cell lines. RT-PCR analysis confirmed that PKG2 is expressed in normal colon mucosa, and although there was no difference in colon tumors, it was undetectable in established colon cancer cell lines. Ectopic expression of PKG2 in LS174T and HT29 cells decreased colony formation in clonogenic assays. This effect of PKG2 on colon cancer cells was not due reduced cell viability but was associated with an increase in G1 arrested cells and increased expression of the differentiation markers CDX2 and MUC2. Consistent with an anti-proliferative and pro-differentiation role of PKG2 in the colon, we observed increased proliferation and larger crypt size in PKG2-/- mice compared to wild type siblings. In addition, there were significantly less mature goblet cells in PKG2-/- mice relative to controls when measured by histological staining with Alcian blue or by immunostaining with anti-MUC2 antibodies. Recent reports suggest that PKG2 might affect differentiation by regulating the transcriptional repressor Sox9. We found that PKG2 could phosphorylate Sox9 at Ser181 in both colon cancer cell lines and in the mouse colon at a position 2/3rd into the crypt. In the colon cancer cell lines, phosphorylation of Sox9 by PKG2 did not affect expression or nuclear localization, and was not sufficient for the regulation of MUC2 or CDX2 expression. The work presented here demonstrates a novel anti-proliferative and pro-differentiation role for PKG2 in the colon, which has an important function in tissue homeostasis. The ability of PKG2 to reduce growth and increase expression of MUC2 and CDX2 tumor suppressors suggests that PKG2 may have therapeutic significance in colon cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3830. doi:10.1158/1538-7445.AM2011-3830
Abstract 3832: Defining molecular mechanisms controlling FES activation and microtubule binding as a potential strategy to limit mast cell recruitment to tumors via the SCF/KIT axis
Authors: Kwok Ester , Allingham John , Craig Andrew W. B.
Published: June 2012

FES protein-tyrosine kinase (PTK) is activated downstream of the KIT receptor in mast cells, and promotes mast cell migration towards Stem cell factor (SCF), the ligand for KIT. Recently, tumor-derived SCF has been implicated in promoting mast cell recruitment leading to increased tumor vascularity and poor prognosis. FES is also a potential therapeutic target in leukemias driven by oncogenic KIT receptors that are constitutively active. Thus, defining FES activation and signaling mechanisms could lead to development of FES inhibitors as potential therapeutics in mast cell-involved tumors or KIT-driven leukemias. FES was previously shown to phosphorylate Tubulin, and localize to microtubules (MTs) in an SH2 domain-dependent manner. We hypothesized that defects in MT reorganization in FES-deficient mast cells may account for the observed defects in migration towards SCF. To map potential MT binding site(s) in FES, we performed MT binding assays, and identified two modes of FES binding, one via the N-terminal F-BAR/FX domains, and also via the SH2 domain. Interestingly, mutations predicted to disrupt oligomerization of the F-BAR/FX domains enhanced MT binding in vitro, and MT localization in vivo. Using in vitro kinase assays, we show that FES preferentially phosphorylates soluble tubulin compared to MTs, suggesting that FES recruitment to MTs may depend on their phosphorylation by other PTKs (such as Src family PTKs). In mast cells treated with SCF, FES co-localizes with MTs, including some localization to the MT organizing center (MTOC). We are currently comparing MTOC orientation in polarized wild-type and FES-deficient mast cells, and examining MT subsets marked by γ-tubulin, acetylation, and detyrosination. In conclusion, we find that FES interacts directly with MTs in vitro, and co-localizes with MTs in vivo, and hypothesize that disruption of FES-MT interaction and/or activation will be relevant therapeutic targets to limit mast cell recruitment in solid tumors, and growth of KIT-driven leukemias.

Funded by an operating grant from Canadian Institutes for Health Research (MOP82882) to AWBC.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3832. doi:10.1158/1538-7445.AM2011-3832
Abstract 3835: Multiple stimulants target different phosphoinositide 3-kinase (PI3K) classes in breast cancer cell lines
Authors: Ehrmantrout Kimberly K. , Thompson James E. , Branch Angie M.
Published: June 2012

Phosphoinositides (PIPs) are an integral part of multiple cell signaling pathways and are metabolized by kinases, phosphatases, and phospholipases. PIPs produced by the activities of phosphoinositide 3-kinase (PI3K) serve as second messengers in cell survival, growth, motility, immune response, inflammation, and apoptosis. Conversely, abnormal production of these signaling lipids leads to proliferative, metabolic, and inflammatory disorders. It is critical to know the absolute and relative concentration of individual PIPs in cell samples, however this analysis is difficult due to their low abundance and the inherent physical properties of lipids. Experiments to measure PIPs have involved radiolabeling cells, extraction of radioactive products, and separation using thin-layer chromatography. The amount of PIPn extracted from cell lysates was determined by means of a standard competitive ELISA format, eliminating the need for radioactivity. Using specific antibodies and binding proteins we developed assays able to differentiate closely related PIPs. These nonradioactive assays employ synthetic di-C16 lipids as standards and are able to measure as little as 0.1 pmol of lipid. Cell-extracted PIPs were added as competitors in these quantitative ELISAs. We used the human breast cancer cell lines MDA-MB-231 and MDA-MB-468 (PTEN deficient) as a model system to evaluate multiple activators of PI3K pathways by looking at levels of PI(3)P, PI(3,4)P2 and PI(3,4,5)P3. Using growth factors, hyperosmolarity, and oxidative stress we found that different stimulants targeted different classes of PI3K. All three classes of PI3K were more active in PTEN deficient cell line (468 cells) than PTEN+/+ (231 cells). We found that all stimulants used increased PI3K products. Pre-incubation with wortmannin, a known PI3K inhibitor, decreased PIPn levels. We propose that hyperactivation of class I and class II PI3K in the absence of PTEN regulation (468 cells) leads to excessive 3’ phosphorylated phosphoinositide levels that are known to activate Akt, We expect the ability to quantify individual phosphoinositides from cells will help assign specific biological functions to these important lipid second messengers.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3835. doi:10.1158/1538-7445.AM2011-3835
Abstract 3838: Mitogen-activated protein kinase phosphatase-1 deficiency increases susceptibility to 4-nitroquinoline 1-oxide-induced neoplasia in murine squamous oral epithelium
Authors: Zhang Xiaoyi , Yu Hong , Kirkwood Keith L.
Published: June 2012

Background: Cytokines and pro-inflammatory factors have critical roles in shaping the malignant transformation that gives rise to head and neck squamous cell carcinoma (HNSCC). Mitogen-activated protein kinases (MAPKs), such as p38, JNK, and ERK, relay information from extracellular signals to control the diverse cellular processes of carcinogenesis. These kinases are negatively regulated by MAPK phosphatases (MKPs), the founding member being mitogen-activated protein kinase phosphatase-1 (MKP-1). Initial studies demonstrated significant over-expression of MKP-1 in human epithelial tumors only in the early phases of disease with levels falling progressively in tumors of higher histological grade and in metastases. We hypothesize low levels of MKP-1 in the HNSCC tumor microenvironment generate high levels of inflammation which promote tumor development and progression.

Methods: To address how regulation of inflammatory cytokine expression via MKP-1 activity may affect HNSCC development and progression, we examined the effects of MKP-1 deficiency on a well characterized model for oral squamous cell carcinoma (OSCC), a subset of HNSCC. MKP-1-/- and wild-type mice were treated with 4-nitroquinoline 1-oxide (4NQO), a surrogate for tobacco exposure, in drinking water for 16 weeks and monitored by oral examination for an additional 16 weeks. Tumor size was assessed by volumetric measurement. Histological grading of the tumors and inflammation were performed in a blinded manner by two collaborating pathologists.

Results: Our studies demonstrated significantly earlier onset of 4NQO-induced OSCC in MKP-1-/- mice compared with wild-type controls. Tumor size and histological grade were found to be more severe in the MKP-1-/- mice. In addition, tissues surrounding the tumors demonstrated a significantly higher degree of inflammation in MKP-1-/- mice. Remarkably, development of esophageal squamous carcinomas was dramatically enhanced in MKP-1-/- mice treated with 4NQO.

Conclusion: These studies demonstrate increased susceptibility of MKP-1-/- mice to 4NQO-induced neoplasia, suggesting an important role for MKP-1 as a negative regulator of HNSCC development. Further work will be done to characterize mechanisms of regulation by assessing levels of MAPK activity and characterizing the pro- and anti-inflammatory milieu of the tumor tissues and supportive stroma. Elucidating the regulation of inflammatory mediators in the tumor microenvironment may reveal critical tumor suppressive interactions mediated by MKP-1 which are lost during cancer initiation and progression.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3838. doi:10.1158/1538-7445.AM2011-3838
Abstract 3844: K-Ras is required for maintaining survivin protein stability in human cancer cells harboring mutant but not wild type K-Ras
Authors: Tecleab Awet , Sebti Said M.
Published: June 2012

The anti-apoptotic protein survivin is maintained at high levels in cancer cells relative to normal cells. The mechanism by which this occurs is ill-understood. Here we show in several human cancer cell lines including those derived from pancreatic, lung and ovarian cancers that knocking down K-Ras expression by siRNA results in decreased survivin levels in cancer cells harboring mutant but not wild type K-Ras. K-Ras knockdown decreases survivin mRNA and protein levels by 50% and 85%, respectively, suggesting that K-Ras regulates survivin transcription as well as protein stability. Consistent with this K-Ras knockdown decreases the levels of ectopically-expressed Myc-tagged survivin. Further support for the ability of K-Ras to regulate the protein stability of survivin is also supported by the fact that the proteasome inhibitor Velcade partially inhibits K-Ras siRNA from decreasing the levels of survivin. Taken together, the data suggest that K-Ras is a contributor to the maintenance of high levels of survivin in human cancer cells which harbor mutant but not wild type K-Ras.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3844. doi:10.1158/1538-7445.AM2011-3844
Abstract 3873: Genetic and epigenetic determinants of outcome in locally advanced head-and-neck cancer
Authors: Nigro Cristiana Lo , Gasco Milena , Vivenza Daniela , Lattanzio Laura , Monteverde Martino , Comino Alberto , Syed Nelofer , Crook Tim , Merlano Marco
Published: June 2012

Despite improved outcome, optimal protocols of combined modality treatments have not been identified in advanced Head and Neck squamous cell carcinoma (HNSCC). Nor has it been established which subgroup of patients will benefit from integrated treatments. Because of the significant treatment-related toxicity, it is critical to design treatments according to molecular predictors in order to maximise therapeutic anti-tumour effect and minimise toxicity. We have investigated the potential utility of candidate predictive biomarkers in a large, homogenous cohort of patients treated in our centre.

One-hundred-eighty-three paraffin-embedded specimens were obtained from 160 patients (136 males and 24 females, median age 0 58 years) with stage III-IV, locally advanced, unresectable HNSCC collected at S. Croce General Hospital since1999. All patients were treated with radical, cisplatin-based, chemo-radiotherapy regimens. P53 mutations in exons 4-10, polymorphism P72R in p53 gene and MDM2 single nucleotide polymorphism (SNP) 309 were analysed by sequencing and pyrosequencing. EGFR expression and HPV status were determined by ICH. Methylation in the CpG island of Dab-2 and DUPS2 was analyzed both by MSP and pyrosequencing.

We previously showed that a functional SNP in the apoptosis signalling domain of p53 influences clinical response of HNSCC to chemotherapy. We now have investigated the hypothesis that, in addition to molecular genetic determinants, methylation profiling can identify distinct subtypes of HNSCC.

p53 mutations were found in 48.5% of samples. Distribution of alleles at codon 72 in p53 was as follows: RR = 50%, RP = 40%, PP = 10%. Distribution of alleles MDM2 SNP309 was: TT =35%, TG = 43%, GG = 22%. Methylation in the Dab-2 and DUPS2 CpG islands was present in 35% and 31%, respectively.

We are currently correlating these data with objective response rate, progression-free survival and overall survival. The final statistical analysis will be presented.

We report the incidence of mutations, SNPs and epigenetic profiling in specific genes in HNSCC. We shall identify correlations with clinical outcome parameters.

The project was partially supported by AIRC grant to M.G.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3873. doi:10.1158/1538-7445.AM2011-3873
Abstract 3877: FTSJ2, a putative ribosomal RNA methyltransferase, suppressed cell invasion and migration in cancer cell line
Authors: Lai Cheng-Wei , Lin Ken-Yo , Liou Fang-Jiue , Chen Hsiao-Ling , Cheng Ju-Chien , Chen Chuan-Mu
Published: June 2012

The E. coli heat shock protein which is called ribosomal RNA large subunit methyltransferase J (RrmJ) responsible for the 2’-O-ribose methylation of A-loop U2552 in the 23S rRNA. This gene is highly conserved from Prokaryota to Eukaryota. In human, three closed homologues of RrmJ have been identified and designated as FTSJ1, FTSJ2 and FTSJ3. In this study, we characterized mammalian FTSJ2 from the amino acid sequences alignment to the mitochondrial localization, and suggested the similar function to RrmJ. The RT-PCR results showed that there were much lower FTSJ2 mRNA expression in eight hepatoma cell lines (HepG2, Hep3B, HHCM, Huh7, 22T, 59T, SK-Hep1, PLC/PRF/5) and one medulloblastoma cell line (Te671), compared with the expression level in normal liver tissue. Subsequently, after the medulloblastoma cells were transfected with FTSJ2 overexpression vector, the FTSJ2-overexpressed Te671 cells (FTSJ2/Te671) showed the inhibition effects of cell migration and invasion in vitro. In addition, this also caused the rearrangement of F-action in FTSJ2/Te671 detected by immunofluorescence. However, recent study showed that FTSJ2 may involve in tumourigenesis, and FTSJ2 gene was activated in non-small cell lung cancer. Thus, we proposed that FTSJ2 may play different roles in different tissues during tumourigenesis through the catalysis of ribosomal RNA methylation in mitochondria.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3877. doi:10.1158/1538-7445.AM2011-3877
Abstract 3881: Genetic alterations in intrahepatic cholangiocarcinoma- revealed by array CGH, SKY and gene expression microarray
Authors: Weng Wen-Hui , Tsao Lee-Cheng , Pang Yeu-Jye , Pang See-Tong , Yeh Chun-Nan
Published: June 2012

Cholangiocarcinoma (CCA) is an adenocarcinoma of the biliary tract, which is considered to be an incurable and rapidly lethal disease. A major problem with CCA surveillance is the lack of reliable biomarkers, therefore, still less no significant aspects of client-centered therapy in clinical treatment so far. Here, we established a 25th week thioacetamide (TAA)-induced cholangiocarcinoma rat cell line model named Chang Gung CCA (CGCCA), aim to figure out the early alterations of cytogenetics, molecular and protein expressed characteristics of CCA. Array comparative genomic hybridization (aCGH) and Spectral karyotyping (SKY), gene expression microarray and immunohistochemistry were performed on either rat CCA tissues or CGCCA cell line in this study. Our results showed complicated alterations of genetic gain or loss on different chromosomal regions could be observed. The data compared with the gene expression profile, thirty-three genes aberrations could be further identified; for example, tumor metastasis-related genes ANXA1 and CLCA3, the genes MMP7 and MMP12 are related with tumor invasion, and proliferation-related genes PKM2 and OSMR have been pinpointed; The number of chromosome ranging between 50 to 56 diplody (2n); despite the apparently high incidence of chromosomal translocation was revealed by cytogenetic analyses, ring and/or giant rod marker chromosomes were detected in almost every CGCCA cells, and the genetic materials are mainly included chromosomes 4 and 8. Herein, we concluded ANXA1, CLCA3, MMP7, MMP12, PKM2 and OSMR genes could be considered as potential biomarkers for the risk of disease. Supernumerary ring or giant rod marker chromosomes could also be considered as a characteristic may be involved in the late stage of CCA. However, the genes involved in the tumorigenesis should be further investigated the function of the genes. In order to verify the clinical significance and potentially to be as therapeutic targets in the future, to confirm onto a series of human CCA tissues are necessary.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3881. doi:10.1158/1538-7445.AM2011-3881
Abstract 3882: The comparison of genomic instability between epidermal growth factor receptor(EGFR) mutation status of lung adenocarcinoma
Authors: Lai Yi-Ling , Yuan Shin-Sheng , Chen Hsuan-Yu , Yu Sung-Liang , Li Ker-Chau , Yang Pan-Chyr
Published: June 2012

We investigated how different the patients of lung adenocarcinoma with and without EGFR activating mutation are in the genomes of their cancer cells. We conducted EGFR mutation test for 141 patients and used high-density even-coverage CGH arrays to chart the sites of DNA copy number alteration (CNA) in primary adenocarcinomas of each patient. We analyzed the site distribution of the most notable alterations from various perspectives and revealed several leading characteristics that converged on chromosome 7p. The clinical relevance of our findings was confirmed by conducting a survival study on an independent group of 114 patients, wherein a gene-signature that used the average copy numbers of 6 genes on 7p as measured by genomic real-time rt-PCR, showed statistical significance in predicting the overall survival and the relapse-free survival for the EGFR-mutation patients but not for the EGFR-wild-type patients. Our findings suggest that chromosome 7p contains very rich information for deciphering the pivotal genomic component contributing to the heterogeneity of lung adenocarcinoma.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3882. doi:10.1158/1538-7445.AM2011-3882