bluuebunny/crossref_metadata_2025 huggingface dataset:
MYC, TP53, and Chromosome 17 Copy‐Number Alterations in Multiple Gastric Cancer Cell Lines and in Their Parental Primary Tumors
Authors: Leal Mariana Ferreira , Calcagno Danielle Queiroz , Borges da Costa Joana de Fátima Ferreira , Silva Tanielly Cristina Raiol , Khayat André Salim , Chen Elizabeth Suchi , Assumpção Paulo Pimentel , de Arruda Cardoso Smith Marília , Burbano Rommel Rodríguez
Published: February 2011

We evaluated whether MYC, TP53, and chromosome 17 copy‐number alterations occur in ACP02, ACP03, and AGP01 gastric cancer cell lines and in their tumor counterpart. Fluorescence in situ hybridization for MYC and TP53 genes and for chromosome 17 was applied in the 6th, 12th, 60th, and 85th passages of the cell lines and in their parental primary tumors. We observed that three and four MYC signals were the most common alterations in gastric cell lines and tumors. ACP02 presented cells with two copies of chr17 and loss of one copy of TP53 more frequently than ACP03 and AGP01. Only ACP03 and AGP01 presented clonal chr17 trisomy with three or two TP53 copies. The frequency of MYC gain, TP53 loss, and chromosome 17 trisomy seems to increase in gastric cell lines compared to their parental tumors. Our findings reveal that these cell lines retain, in vitro, the genetic alterations presented in their parental primary tumors.
In Situ Generated Colloid Transport of Cu and Zn in Reclaimed Mine Soil Profiles Associated with Biosolids Application
Authors: Miller Jarrod O. , Karathanasis Anastasios D. , Matocha Christopher J.
Published: February 2011

Areas reclaimed for agricultural uses following coal mining often receive biosolids applications to increase organic matter and fertility. Transport of heavy metals within these soils may be enhanced by the additional presence of biosolids colloids. Intact monoliths from reclaimed and undisturbed soils in Virginia and Kentucky were leached to observe Cu and Zn mobility with and without biosolids application. Transport of Cu and Zn was observed in both solution and colloid associated phases in reclaimed and undisturbed forest soils, where the presence of unweathered spoil material and biosolids amendments contributed to higher metal release in solution fractions. Up to 81% of mobile Cu was associated with the colloid fraction, particularly when gibbsite was present, while only up to 18% of mobile Zn was associated with the colloid fraction. The colloid bound Cu was exchangeable by ammonium acetate, suggesting that it will release into groundwater resources.
Gene Expression Differences between Enriched Normal and Chronic Myelogenous Leukemia Quiescent Stem/Progenitor Cells and Correlations with Biological Abnormalities
Authors: Affer M. , Dao S. , Liu C. , Olshen A. B. , Mo Q. , Viale A. , Lambek C. L. , Marr T. G. , Clarkson B. D.
Published: February 2011

In comparing gene expression of normal and CML CD34+ quiescent (G0) cell, 292 genes were downregulated and 192 genes upregulated in the CML/G0 Cells. The differentially expressed genes were grouped according to their reported functions, and correlations were sought with biological differences previously observed between the same groups. The most relevant findings include the following. (i) CML G0 cells are in a more advanced stage of development and more poised to proliferate than normal G0 cells. (ii) When CML G0 cells are stimulated to proliferate, they differentiate and mature more rapidly than normal counterpart. (iii) Whereas normal G0 cells form only granulocyte/monocyte colonies when stimulated by cytokines, CML G0 cells form a combination of the above and erythroid clusters and colonies. (iv) Prominin-1 is the gene most downregulated in CML G0 cells, and this appears to be associated with the spontaneous formation of erythroid colonies by CML progenitors without EPO.
Phaeochromocytoma Mimicking Scleroderma
Authors: Khoo Joan Joo-Ching , Pratt Edward John
Published: February 2011

We describe a patient with adrenal phaeochromocytoma who presented with peripheral vasospasm and ischaemia and other manifestations mimicking features of systemic sclerosis, which resolved after resection of the tumor. Phaeochromocytoma should be suspected in patients with features of scleroderma who are negative for auto-antibodies.
Cyclin D1 Inhibits Mitochondrial Activity in B Cells
Authors: Tchakarska Guergana , Roussel Mikel , Troussard Xavier , Sola Brigitte
Published: February 2011

Cyclin D1 is a cell cycle regulatory protein that acts at the G1-S transition, following its binding to and activation by the cyclin-dependent kinases 4 or 6. Cyclin D1 is absent from the entire B-cell lineage but is present in a large percentage of 2 types of malignant B-cell hemopathy—mantle cell lymphoma and multiple myeloma— suggesting a major role of this protein in the malignancy process. We show here, in an experimental model of cyclin D1 fusion protein transduction in mature B cells, that, cyclin D1 inhibits total mitochondrial activity. Cyclin D1 is localized at the outer mitochondrial membrane, bound to a voltage-dependent anion channel through its central domain, and it competes with hexokinase 2 for binding to this channel. The bound cyclin D1 decreases the supply of ADP, ATP, and metabolites, thereby reducing energy production. This function of cyclin D1 was also reported by others in normal and transformed mammary epithelial cells, suggesting that it may be ubiquitous. Cancer Res; 71(5); 1690–9. ©2011 AACR.
CNT1 Expression Influences Proliferation and Chemosensitivity in Drug-Resistant Pancreatic Cancer Cells
Authors: Bhutia Yangzom D. , Hung Sau Wai , Patel Bhavi , Lovin Dylan , Govindarajan Rajgopal
Published: February 2011

Overcoming the inherent chemoresistance of pancreatic cancers remains a major goal of therapeutic investigations in this disease. In this study, we discovered a role for the human concentrative nucleoside transporter-1 (hCNT1; SLC28A1), a high-affinity pyrimidine nucleoside transporter, in determining the chemosensitivity of human pancreatic cancer cells to gemcitabine, the drug used presently as a standard of care. Compared with normal pancreas and pancreatic ductal epithelial cells, hCNT1 expression was frequently reduced in pancreatic tumors and tumor cell lines. In addition, hCNT1-mediated 3H-gemcitabine transport was lower in pancreatic cancer cell lines and correlated with cytotoxic IC50 estimations of gemcitabine. In contrast to gemcitabine-sensitive pancreatic cancer cell lines, MIA PaCa-2, a gemcitabine-resistant pancreatic cancer cell line, exhibited relatively restrictive, cell cycle-dependent hCNT1 expression and transport. hCNT1 translation was suppressed in the late G1-enriched MIA PaCa-2 cell population possibly in an miRNA-dependent manner, which corresponded with the lowest hCNT1-mediated gemcitabine transport during this phase. Although hCNT1 protein was induced during G1/S transition, increased hCNT1 trafficking resulted in maximal cell surface recruitment and transport-overshoot in the G2/M phase-enriched cell population. hCNT1 protein was directed predominantly to proteasomal or lysosomal degradation in S or G2/M phase MIA PaCa-2 cells, respectively. Pharmacological inhibition of hCNT1 degradation moderately increased cell surface hCNT1 expression and cellular gemcitabine transport in MIA PaCa-2 cells. Constitutive hCNT1 expression reduced clonogenic survival of MIA PaCa-2 cells and steeply augmented gemcitabine transport and chemosensitization. In addition to supporting a putative tumor suppressor role for hCNT1, our findings identify hCNT1 as a potential candidate to render drug-resistant pancreatic cancer cells amenable to chemotherapy. Cancer Res; 71(5); 1825–35. ©2011 AACR.
Steroid Receptor Coactivator-1 Upregulates Integrin α5 Expression to Promote Breast Cancer Cell Adhesion and Migration
Authors: Qin Li , Chen Xian , Wu Yelin , Feng Zhen , He Tao , Wang Li , Liao Lan , Xu Jianming
Published: February 2011

Metastatic breast cancer remains a lethal disease with poorly understood molecular mechanisms. Steroid receptor coactivator-1 (SRC-1 or NCOA1) is overexpressed in a subset of breast cancers with poor prognosis. It potentiates gene expression by serving as a coactivator for nuclear receptors and other transcription factors. We previously reported that SRC-1 promotes breast cancer metastasis without affecting primary mammary tumor formation. Herein, we found that SRC-1 deficiency in mouse and human breast cancer cells substantially reduced cell adhesion and migration capabilities on fibronectin and significantly extended the time of focal adhesion disassembly and reassembly. In agreement with this phenotype, SRC-1 expression positively correlated with integrin α5 (ITGA5) expression in estrogen receptor–negative breast tumors whereas SRC-1 deficiency decreased ITGA5 expression. Furthermore, ITGA5 reduction in SRC-1–deficient/insufficient breast cancer cells or knockdown of ITGA5 in SRC-1–expressing breast cancer cells was associated with a disturbed integrin-mediated signaling. Critical downstream changes included reduced phosphorylation and/or dampened activation of focal adhesion kinase, paxillin, Rac1, and Erk1/2 during cell adhesion. Finally, we found that SRC-1 enhanced ITGA5 promoter activity through an AP-1 (activator protein)–binding site proximal to the transcriptional initiation site; both SRC-1 and c-Jun were recruited to this promoter region in breast cancer cells. These results show that SRC-1 can promote breast cancer metastasis by directly enhancing ITGA5 expression and thus promoting ITGA5-mediated cell adhesion and migration. Therefore, targeting ITGA5 in SRC-1–positive breast cancers may result in inhibition of SRC-1–promoted breast cancer metastasis. Cancer Res; 71(5); 1742–51. ©2011 AACR.
Hypoxia Predicts Aggressive Growth and Spontaneous Metastasis Formation from Orthotopically Grown Primary Xenografts of Human Pancreatic Cancer
Authors: Chang Qing , Jurisica Igor , Do Trevor , Hedley David W.
Published: February 2011

Hypoxia in solid tumors is associated with treatment resistance and increased metastatic potential. Although hypoxia has been reported in pancreatic cancer patients, there is little direct evidence that this contributes to their overall poor prognosis. To address this, we examined the associations between hypoxia and biological aggression in a series of patient-derived xenografts grown orthotopically. Early passage xenografts were established from 16 patients undergoing surgery for pancreatic cancer and maintained in the pancreas of immune-deprived mice. Hypoxic cells were labeled using the 2-nitroimidazole probe EF5 and stained for immunofluorescence microscopy of tissue sections or as cell suspensions for flow cytometry. Bromodeoxyuridine (BrdUrd) uptake, microvessel density, cleaved caspase-3, and the differentiation markers E-cadherin, cytokeratin 19, and vimentin were analyzed in relation to hypoxia. Orthotopic implants closely resembled the histology of the original surgical samples. The 16 primary xenografts showed a wide range in their growth rates and metastatic potential, reminiscent of the spectrum of behavior seen in the clinic. EF5 labeling, tumor growth rates, and metastatic patterns were highly consistent within replicates, indicating a significant transmissible (genetic or epigenetic) component. Hypoxia was highly correlated with rapid tumor growth, increased BrdUrd uptake, and with spontaneous metastasis formation. mRNA expression analysis showed increased expression of genes involved in cell survival and proliferation in the hypoxic models. The results suggest that hypoxia is a major adverse prognostic factor in pancreatic cancer patients and support the introduction of techniques to measure hypoxia directly in patients and the development of treatment protocols to target hypoxia. Cancer Res; 71(8); 3110–20. ©2011 AACR.
Prostate Cancer Predisposition Loci and Risk of Metastatic Disease and Prostate Cancer Recurrence
Authors: Ahn Jiyoung , Kibel Adam S. , Park Jong Y. , Rebbeck Timothy R. , Rennert Hanna , Stanford Janet L. , Ostrander Elaine A. , Chanock Stephen , Wang Ming-Hsi , Mittal Rama D. , Isaacs William B. , Platz Elizabeth A. , Hayes Richard B.
Published: February 2011

Purpose: Genome-wide association studies (GWAS) have identified multiple novel prostate cancer predisposition loci. Whether these common genetic variants are associated with incident metastatic prostate cancer or with recurrence after surgical treatment for clinically localized prostate cancer is uncertain.

Experimental Design: Twelve single nucleotide polymorphisms (SNPs) were selected for study in relation to prostate metastatic cancer and recurrence, based on their genome-wide association with prostate cancer in the Cancer Genetic Markers of Susceptibility (CGEMS). To assess risk for metastatic disease, we compared genotypes for the 12 SNPs by logistic regression of 470 incident metastatic prostate cancer cases and 1,945 controls in 3 case-control studies. To assess the relationship of these SNPs to risk for prostate cancer recurrence, we used Cox regression in a cohort of 1,412 men treated for localized prostate cancer, including 328 recurrences, and used logistic regression in a case-case study, comparing 450 recurrent versus 450 nonrecurrent prostate cancer cases. Study-specific relative risks (RRs) for risk of metastatic disease and recurrence were summarized using meta-analysis, with inverse variance weights.

Results:MSMB rs10993994 (per variant allele summary RR = 1.24, 95% CI = 1.05–1.48), 8q24 rs4242382 (RR = 1.40, 95% CI = 1.13–1.75), and 8q24 rs6983267 (RR = 0.67, 95% CI = 0.50–0.89) were associated with risk for metastatic prostate cancer. None of the 12 SNPs was associated with prostate cancer recurrence.

Conclusions: SNPs in MSMB and 8q24 which predispose to prostate cancer overall are associated with risk for metastatic prostate cancer, the most lethal form of this disease. SNPs predictive of prostate cancer recurrence were not identified, among the predisposition SNPs. GWAS specific to these 2 phenotypes may identify additional phenotype-specific genetic determinants. Clin Cancer Res; 17(5); 1075–81. ©2011 AACR.
Pancreatic Tumor Suppression by Benzyl Isothiocyanate Is Associated with Inhibition of PI3K/AKT/FOXO Pathway
Authors: Boreddy Srinivas Reddy , Pramanik Kartick C. , Srivastava Sanjay K.
Published: February 2011

Purpose: Our previous studies have shown that benzyl isothiocyanate (BITC) suppress pancreatic cancer growth by inducing apoptosis but the molecular mechanism was unclear. In this study we hypothesized the involvement of PI3K/AKT/FOXO pathway in BITC-induced apoptosis.

Experimental Design: Mice were implanted BxPC-3 tumor xenografts and orally gavaged with 12 μmol BITC. Plasma and tumor BITC concentration was estimated by liquid chromatography/tandem mass spectrometry. BxPC-3 and PanC-1 cells were used to elucidate PI3K/AKT/FOXO pathway. Electrophoretic mobility shift assay (EMSA), DNA binding activity, immunofluorescence, and gene transfection were used to delineate the mechanism.

Results: BITC-treated mice showed 43% less tumor growth as compared with control mice and correlated well with the therapeutic concentrations of 6.5 μmol/L BITC achieved in plasma and 7.5 μmol/g BITC in tumor tissue. Western blot analyses and immunohistochemistry revealed that tumors from BITC-treated mice showed reduced phosphorylation of PI3K, AKT, PDK1, mTOR, FOXO1, and FOXO3a and increased apoptosis. Complementing our in vivo results, we made similar observations in a dose- and time-dependent manner in BITC-treated BxPC-3 and Panc-1 cells. Binding of FOXO1 with 14-3-3 proteins was also reduced drastically by BITC treatment indicating nuclear retention of FOXO1 and this observation was further confirmed with EMSA, immunofluorescence, DNA binding, and upregulation of FOXO-responsive proteins Bim, p27, and p21 in BxPC-3 cells. Overexpression of AKT by transient transfection significantly blocked the modulation of FOXO proteins and protected the cells from BITC-mediated apoptosis and growth suppression.

Conclusions: Our results provide convincing evidence on the involvement of PI3K/AKT/FOXO pathway in BITC-mediated pancreatic tumor growth suppression. Clin Cancer Res; 17(7); 1784–95. ©2011 AACR.
A Novel HSP90 Inhibitor Delays Castrate-Resistant Prostate Cancer without Altering Serum PSA Levels and Inhibits Osteoclastogenesis
Authors: Lamoureux Francois , Thomas Christian , Yin Min-Jean , Kuruma Hidetoshi , Fazli Ladan , Gleave Martin E. , Zoubeidi Amina
Published: February 2011

Purpose: Prostate cancer responds initially to antiandrogen therapies; however, progression to castration-resistant disease frequently occurs. Therefore, there is an urgent need for novel therapeutic agents that can prevent the emergence of castrate-resistant prostate cancer (CRPC). HSP90 is a molecular chaperone involved in the stability of many client proteins including Akt and androgen receptor (AR). 17-Allylamino-17-demethoxy-geldanamycin (17-AAG) has been reported to inhibit tumor growth in various cancers; however, it induces tumor progression in the bone microenvironment.

Methods: Cell growth, apoptosis, and AR transactivation were examined by crystal violet assay, flow cytometric, and luciferase assays, respectively. The consequence of HSP90 therapy in vivo was evaluated in LNCaP xenograft model. The consequence of PF-04928473 therapy on bone metastasis was studied using an osteoclastogenesis in vitro assay.

Results: PF-04928473 inhibits cell growth in a panel of prostate cancer cells, induces cell-cycle arrest at sub-G1, and leads to apoptosis and increased caspase-3 activity. These biological events were accompanied by decreased activation of Akt and Erk as well as decreased expression of Her2, and decreased AR expression and activation in vitro. In contrast to 17-AAG, PF-04928473 abrogates RANKL-induced osteoclast differentiation by affecting NF-κB activation and Src phosphorylation. Finally, PF-04929113 inhibited tumor growth and prolonged survival compared with controls. Surprisingly, PF-04929113 did not reduce serum prostate-specific antigen (PSA) levels in vivo; in parallel, these decrease in tumor volume.

Conclusion: These data identify significant anticancer activity of PF-04929113 in CRPC but suggest that serum PSA may not prove useful as pharmacodynamic tool for this drug. Clin Cancer Res; 17(8); 2301–13. ©2011 AACR.
Abstract 1001: Effects of matrix compositions on the development of breast acini and ducts in a 3D in-vitro culture model
Authors: Swamydas Muthulekha , Eddy Jill , Burg Karen L. , Dréau Didier
Published: February 2011

Background: Epithelial cells of the breast respond to cues from the stromal components and develop into polarized and highly organized functional acinar and ductal structures. Extracellular matrix components and increased stiffness in particular have been shown to promote breast cancer. The stroma - cell interactions that determine the cellular organization and differentiation are in part reproduced in a 3D environment. Whether alterations of the microenvironment in 3D cultures could mimic phenotypic alterations observed during breast cancer progression remain to be fully determined. Here we present the effects of various stroma physical compositions on the ability of heterotypic 3D cultures of epithelial and mesenchymal stem cells to organize into acinar and tubular structures.

Material & Methods: The normal murine mammary gland (NMuMG) cells were cultured either alone or in combination (30:70) with mouse mesenchymal stem cells (D1), in 3D cell culture conditions generated by agarose, collagen and Matrigel® alone or a combination thereof. After 3-5 days in culture, cell distribution, organization and the presence of acinus-like and tubule-like structures were determined.

Results: By immunohistochemistry, NMuMG cells co-cultured with D1 cells were shown to form acinar structures with the NMuMG epithelial cells surrounding a lumen composed of dead cells whereas the D1 cells were mostly peripheral. Immuno-staining for laminin indicated the presence of basement membrane when NMuMG cells were grown in Matrigel® alone or co-cultured with D1 cells in a combination of Matrigel® and collagen I. The number of acinar structures was significantly higher in cultures grown in combination of agarose with Matrigel® or collagen I when compared to the cultures grown in Matrigel® or collagen I alone (P<0.05). No tubular structures were formed when agarose was included in the matrix, regardless of the combination. In Matrigel® but not in collagen I/ Matrigel® microenvironment the number of tubular structures was significantly increased in NMuMG /D1 co-culture when compared to culture of NMuMG cells alone (P<0.05).

Conclusion: These results indicate that the physical and biochemical properties of the matrix and cellular composition alter the organization of the mammary gland. Furthermore, they underline the likely critical role of the extracellular matrix in breast cancer progression.

Supported by grants from the Department of Defense Era of Hope Program and the National Science Foundation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1001.
Abstract 1003: Cancer-associated myofibroblast may stimulate vimentin expression of gastric cancer cells
Authors: Yuhiko Fuyuhiro , Yashiro Masakazu , Noda Satoru , Doi Yosuke , Kato Yukihiro , Kashiwagi Shinichiro , Kubo Naoshi , Tanaka Hiroaki , Muguruma Kazuya , Inoue Toru , Yamada Nobuya , Ohira Masaichi , Hirakawa Kosei
Published: February 2011

Introduction: Epithelial-mesenchymal transition (EMT) is a process in which cells undergo a developmental switch from an epithelial to a mesenchymal phenotype. During the EMT, epithelial cells seem to acquire malignant properties, with changes in cell morphology, invasion, and migration capacity. Vimentin is one of EMT markers for cancer cells, and vimentin expression of epithelial cells has been reported to be associated with the malignant phenotype of cancer cells in vitro. Fibroblasts, especially cancer-associated myofibroblasts which express α-smooth muscle actin, may affect the malignant phenotype of cancer cells. Then, we examined the effect of fibroblasts on the vimentin expression of gastric cancer cells.

Materials & Methods: A gastric cancer cell line, OCUM-2MD3, and 3 gastric fibroblasts, CaF1, CaF32, and CaF33 those are derived from gastric tumor, were used in this study. Serum-free conditioned medium (SF-CM) from gastric fibroblasts was collected and stored at −20°C. As a control, DMEM was used instead of SF-CM. Following the addition of SF-CM into OCUM-2MD3 cells for 3 days, vimentin expression of OCUM-2MD3 was analyzed by reverse transcription-PCR. Correlation between vimentin expression of cancer cells and α-smooth muscle actin expression of fibroblasts was analayzed by Pearson's correlation coefficient test.

Results: Vimentin expression of gastric cancer cells was 2.3 fold, 1.1 fold, and 1.3 fold increased by addition of SF-CM from CaF1, CaF32, and CaF33, respectively. α-Smooth muscle actin expression of each fibroblasts from CaF1, CaF32, and CaF33 was 7.7 fold, 1.3 fold, and 2.1 fold higher compared to the normal fibroblasts, respectively. Gastric cancer cells showed EMT by SF-CM. A significant correlation (p=0.017) was found between the α-smooth muscle actin expression level of fibloblast and the vimentin expression level of cancer cells.

Conclusions: Tumor-stromal myofibroblast may stimulate malignant phenotype of gastric cancer cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1003.
Abstract 1013: Hepatic stellate cells promote progression of cholangiocarcinoma in vitro and in vivo
Authors: Okabe Hirohisa , Beppu Toru , Hayashi Hiromitsu , Ishiko Takatoshi , Masuda Toshiro , Otao Ryu , Watanabe Masayuki , Takamori Hiroshi , Baba Hideo
Published: February 2011

Background: Although tumor-stroma interaction has been discussed, the role of hepatic stellate (HS) cells against cancer, especially cholangiocarcinoma, has not been clarified. Although hepatic stellate cells have been known to have a pivotal role in fibrogenesis in liver, the influence of them on liver cancer has been poorly investigated. We previously reported hepatic stellate cells may exist in cancer stroma of intrahepatic cholangiocarcinoma and interact with cancer cells in vitro. The aim of this study was to investigate the effect of HS cells on cholangiocarcinoma cells progression in vitro and in vivo and to identify key proteins which regulate their interaction.

Methods: The effects of cholangiocarcinoma cells’ conditioned medium (CC-CM) on activation and proliferation of HS cells (LI90 and LX-2), the influences of HS cells conditioned medium (HS-CM) on in vitro proliferation and invasion of cholangiocarcinoma cells (HuCCT-1 and RBE), and the effects of their interaction on human umbilical venous endothelial cells (HUVECs) tube formation were assessed using each of conditioned medium. The effect of HS cells on tumor growth was examined in vivo by subcutaneous co-injection of them. To clarify the factors inducing their interaction, cytokine array was performed to assess the secreted proteins induced by co-culture of cholangiocarcinoma and HS cells.

Results: CC-CM activated HS cells and increased their proliferation. HS-CM dose-dependently increased cholangiocarcinoma cell proliferation and invasion. Chemotherapy to cholangiocarcinoma cells was less effective treated with HS-CM. HS-CM activated the mitogen-activated protein kinase (MAPK) and Akt pathways in tumor cells. The indirect interaction of cholangiocarcinoma and HS cells promote the tube formation of HUVECs. Subcutaneous co-injection of tumor cells with HS cells in nude mouse resulted in increased tumor size and weight. There found to be several proteins including interleukin-1β (IL1β) that remarkably increased by co-culture of them in culture medium and they were thought to be key proteins which regulated tumor-stromal interaction.

Conclusions: This study indicated that HS cells have an important role in accelerating cholangiocarcinoma progression. Several proteins including IL1β induced by co-culture model may be therapeutic targets which focus tumor-stromal interaction in cholangiocarcinoma.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1013.
Abstract 1028: Regulation of glucocorticoid-evoked apoptosis in human leukemic CEM cells by the C. elegans ces2 ortholog E4BP4
Authors: Jessica Beach A. , Medh Rheem D.
Published: February 2011

Identification of regulatory gene pathways is crucial for fully understanding apoptosis in human cells. In C. elegans, motorneuron apoptosis is regulated via the ces2→ces1→egl1 pathway. We propose that an analogous pathway exists in humans. It has been confirmed that the ces2 ortholog E4BP4 and the egl1 orthologs bim/puma are regulators in glucocorticoid (GC)-evoked apoptosis in human lymphoid leukemic CEM cells. We aim to determine whether E4BP4 regulates bim/puma in a manner similar to ces2's regulation of apoptosis via the down regulation of the ces1 ortholog slug/snail. In this study, E4BP4-dependence of slug/snail and bim/puma regulation by GCs was evaluated in CEM sister cell lines. Cells were treated with ethanol (control) or 1 μM Dexamethasone (a synthetic GC) for 24 h for evaluation of transcript and protein levels, and for 96 h for trypan blue exclusion-based viability assays. Relative mRNA expression and protein abundance of bim/puma and slug/snail were evaluated by qRT-PCR and Western analysis, respectively. Both bim/puma and slug/snail promoters were analyzed using promoter scanning software (Transcriptional Regulatory Element Database) to locate potential E4BP4 response elements. GC-evoked apoptosis in sensitive CEM-C7-14 cells correlated with E4BP4 upregulation, and GC-resistance in CEM-C1-15 cells could be overcome by stable ectopic expression of mouse E4BP4 (cell line CEM-C1-15-mE#3). Flow cytometric analysis of DNA content following propidium iodide labeling has confirmed the expected G1 arrest and accumulation of sub-G1 populations in C7-14 and C1-15-mE#3 cells. qRT-PCR and Western blotting results have confirmed differential bim expression; with C7-14 cells showing a significant two-fold increase in comparison to C1-15 cells. qRT-PCR results for puma, slug, and snail did not show significant differences in expression levels between C7-14, C1-15 and C1-15-mE#3 cells. Furthermore, the expected down regulation of slug/snail in the GC-sensitive C7-14 cells was not observed. Our data suggest that in CEM cell apoptosis, E4BP4-dependent downregulation of slug/snail is not an intermediate for bim upregulation. Unlike the ces2→ces1→egl1 pathway in C. elegans, E4BP4 may use alternative pathways (c-myc, c-jun, cyclin D3, etc.) or directly upregulate bim to evoke apoptosis in CEM cells.

JAB is a recipient of a CSUN Graduate Equity Fellowship & a Graduate Fellowship for Outstanding Research Promise in Science & Mathematics from the CSUN College of S&M. Project funding by NIH grant (SC3 GM 081099-01) awarded to RDM.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1028.
Abstract 1030: Arsenic trioxide plus ethacrynic acid-butyl ester is synergistic to induce apoptosis in myeloid leukemia cells through upregulation of NOXA
Authors: Wang Rui , Liu Changda , Waxman Samuel , Jing Yongkui
Published: February 2011

Arsenic trioxide (As2O3) induces complete remission in acute promyelocytic leukemia (APL) patients. However, other types of myeloid leukemia patients are less responsive to As2O3 treatment. APL t(15;17) NB4 cells are more sensitive to As2O3 apoptosis induction than other types of myeloid leukemia cells such as HL-60 and K562 cells. Previously we reported that As2O3 induces apoptosis in NB4 cells through a reactive oxygen species (ROS)-mediated pathway which is attenuated by the expression of glutathione-s-transferase π (GSTπ). NB4 cells contain lower levels of GSTπ than other types of myeloid leukemia cells. Therefore, inhibition of GSTπ should enhance As2O3 induction of apoptosis in other types of myeloid leukemia cells. We investigated the combined effect of As2O3 with ethacrynic acid (EA) and a more potent non-diuretic EA analogue ethacrynic acid-butyl ester (EABE), known GSTπ inhibitors, to induce apoptosis in HL-60 and K562 cells. As2O3 at clinical achievable concentrations (1-2 µM) did not induce apoptosis in HL-60 cells nor in K562 cells. EA at a concentration lower than 60 μM did not induce apoptosis in both cell lines either. As2O3 at 2 μM plus EA at 60 μM are synergistic to induce apoptosis in both HL-60 and K562 cells as measured by Annexin V staining, PARP cleavage and caspase activation. More intriguing, EABE at 1 μM (60 fold lower than EA), which inhibits GSTπ activity, exhibited synergistic induction with As2O3 in K562 cells. Combined index of As2O3 with EABE to induce apoptosis in K562 cells calculated by CompuSyn software was less than 1.0 which indicated synergism. Apoptosis induction by the combination treatment of As2O3 with EABE correlated with the upregulation of the Bcl-2 homology 3 (BH3)-only pro-apoptotic protein NOXA. Knock-down of NOXA using siRNA blocked apoptosis induction of As2O3 plus EABE, while, knock-down of Mcl-1 enhanced the apoptosis induction of As2O3 plus EABE. Immunoprecipiation with Mcl-1 antibody revealed that NOXA bond to Mcl-1 in the K562 cells treated with As2O3 plus EABE. Inhibition of ROS production did not inhibit the upregulation of NOXA nor the apoptosis induction of As2O3 plus EABE treatment. Our data suggest that As2O3 plus EABE induce apoptosis through a novel mechanism which induces NOXA and sequesters Mcl-1 leading to the activation the intrinsic apoptotic pathway.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1030.
Abstract 1040: Most putative BH3 mimetics preferentially antagonize the anti-apoptotic protein MCL-1 rather than BCL-2
Authors: Albershardt Tina C. , Pletnev Alexandre A. , Eastman Alan
Published: February 2011

The anti-apoptotic proteins of the BCL-2 family (BCL-2, BCL-X and MCL-1) have been variously implicated in the resistance of tumors to anti-cancer therapies. Numerous small molecules have been developed to mimic the binding of BH3-only proteins to the BH3 pocket of anti-apoptotic proteins. These “BH3 mimetics” thereby disrupt the ability of anti-apoptotic proteins to bind and inhibit pro-apoptotic proteins. ABT-737 is a BH3 mimetic that inhibits BCL-2 and BCL-X but not MCL-1 and kills cells in a BAX/BAK-dependent manner. The ability of other BH3 mimetics to inhibit anti-apoptotic proteins has been questioned because they kill cells in a BAX/BAK-independent manner. It has thus been assumed that the actions of these BH3 mimetics must be mediated by off-target effects. In the NB4 leukemia cell line, we show for the first time that most BH3 mimetics preferentially inhibit MCL-1. Six putative BH3 mimetics investigated (GX015-070, gossypol, apogossypol, methoxy-antimycin A, HA14-1 and S1) led to an increase in both NOXA and MCL-1. NOXA binds to MCL-1 and targets the NOXA/MCL-1 complex for degradation. Hence, their accumulation is consistent with these BH3 mimetics disrupting the formation of the complex and preventing its degradation. The rapid increase in NOXA observed within 6 h is associated with the induction of apoptosis within 24 h. Considering a recent report that NOXA can induce cell death through a calcium-dependent pathway, our findings suggest that the BAX/BAK-independent cell death induced by BH3 mimetics may in fact be the result of their on-target mechanism of action. ABT-737 did not increase the levels of NOXA or MCL1, which is consistent with its inability to inhibit the binding of MCL-1 to NOXA. Furthermore, ABT-737 was the only BH3 mimetic that disrupted the binding of BCL-2 to BAD, suggesting that the other BH3 mimetics did not inhibit BCL-2. Lastly, gossypol, S1, and HA14-1 at a concentration that increased NOXA but not apoptosis, sensitized NB4 cells to ABT-737, inducing apoptosis within 6 h. We thus conclude that most BH3 mimetics inhibit MCL-1 but not BCL-2 or BCL-X, while combinations can be used to inhibit all these anti-apoptotic proteins and rapidly induce apoptosis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1040.
Abstract 1054: Identification of progesterone receptor membrane component-1 as the putative sigma-2 receptor
Authors: Xu Jinbin , Zeng Chenbo , Chu Wenhua , Pan Fenghui , Mach Robert H.
Published: February 2011

Sigma binding sites represent a unique class of receptors and are highly expressed in tumor cells. Two sigma binding site subtypes, known as sigma-1 and sigma-2 receptors, were distinguished based on differences in their pharmacological profiles and molecular weights. The sigma-1 receptor gene has been cloned and well characterized in the CNS and cancer biology. However, the sigma-2 receptor has not yet been cloned. The purpose of the current study is to identify and clone sigma-2 receptor. We have synthesized an irreversible sigma-2 receptor selective photoaffinity probe, WC-II-21, containing a fluorescein isothiocyanate (FITC) group. This ligand has high binding affinity for sigma-2 receptor (Ki = 13 nM) and relatively low binding affinity for sigma-1 receptor (Ki > 1,000 nM). This ligand irreversibly labeled sigma-2 receptors in the rat liver membrane fractions after UV irradiation. The ligand-labeled proteins were solubilized, enriched and then separated through sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The position of a predominant band of the ligand-labeled protein in the gel was determined by western blot analysis using FITC antibody. MALDI-mass spectrometry analysis for the ligand-labeled proteins indentified the protein progesterone receptor membrane component-1 (PGRMC-1) with a convincingly high score of 81. Based on the similarities of the reported biological characteristics and functions between PGRMC1 and sigma-2 receptor, PGRMC1 was investigated in the current study. Immunocytochemistry revealed that PGRMC1 colocalized with molecular markers of endoplasmic reticulum and mitochondria in human melanoma cell line MDA-MB435. The subcellular localization of sigma-2 receptor also colocalized with fluorescent markers of endoplasmic reticulum and mitochondria as reported previously from our group (Cancer Res 2007; 67, 6708-6716). Thus, PGRMC1 and sigma-2 receptor appear to localize in the same subcellular organelles in the cell. PGRMC1 was knocked down with PGRMC1 siRNA in MDA-MB435 cells. Sigma-2 radioligand binding activity was significantly reduced in the PGRMC1 knocked down cells relative to the nontargeting siRNA transfected cells. These data suggest that PGRMC1 is the putative sigma-2 receptor. Further validation of PGRMC1 as the sigma-2 receptor is ongoing in our group. (Supported by CA 102869).

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1054.
Abstract 1060: Regulation of SDF-1 (CXCL12) production by mesenchymal stromal cells in the tumor microenvironment
Authors: Amiri Sohrab , Lin Siang-Yo , Picinich Sonia , DeRenzo Christopher , Li Jiadong , Banerjee Debabrata , Glod John W.
Published: February 2011

The tumor microenvironment is made up of a number of cell types including fibroblasts, pericytes, endothelial cells, macrophages, and neoplastic cells. The interaction between cellular components of the microenvironment is critical for processes such as tumor growth, angiogenesis, and metastasis. Mesenchymal stromal cells (MSCs) are an important component of the tumor microenvironment with roles in key aspects of tumor progression such as cell growth and metastasis. Stromal-derived factor −1 (SDF-1) has been identified as a key signaling molecule in the interaction between MSCs and neoplastic cells. SDF-1 acting through its cognate receptors CXCR4 and CXCR7 is known to mediate diverse processes such as chemotaxis, angiogenesis, hematopoiesis, metastasis, and tumor growth. In this study we investigated the regulation of SDF-1 production by MSCs.

We show that the tumor suppressor p53 is one of the major regulators of SDF-1 signaling in MSCs within the tumor stroma. We also illustrate that a decrease in p53 function leads to increased SDF-1 production by MSCs which results in increased migration of MSCs. Decreased p53 expression by MSCs leads to increased SDF-1 production with and increased recruitment to the tumor microenvironment. An improved understanding of the molecular mechanisms controlling SDF-1 production by MSCs may identify novel ways of therapeutically targeting tumor - stromal interactions.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1060.
Abstract 1083: Alpha-santalol, a chemopreventive agent against skin cancer, causes G2/M cell cycle arrest in both p53 mutated human epidermoid carcinoma A431 cells, and p53 wild-type human melanoma UACC-62 cells
Authors: Zhang Xiaoying , Chen Wei , Guillermo Ruth , Chandrasekher Gudiseva , Kaushik Radhey S. , Fahmy Hesham , Dwivedi Chandradhar
Published: February 2011

α-Santalol, an active component of sandalwood oil, has shown chemopreventive effects on skin cancer development in murine models by either chemical- or UVB-induced skin carcinogenesis. α-Santalol has shown induction of apoptosis in both murine models and epidermoid carcinoma A431 cells. However, to the best of our knowledge, effects of α-santalol on cell cycle have not been studied. Thus, the objective of this study was to investigate the effects of α-santalol on cell cycle progression and protein components involved in the regulation of cell cycle in both p53 mutated human epidermoid carcinoma A431 cells and p53 wide-type human melanoma UACC-62 cells to elucidate the mechanism(s) of action.

MTT assay was used to determine cell viability in A431 cells, UACC-62 cells and monkey kidney CV-1 cells; fluorescence-activated cell sorting (FACS) analysis of propidium iodide staining was used for determining cell cycle distribution in A431 cells and UACC-62 cells, and Western immunoblotting was used for determining the expression of various proteins involved in the cell cycle progression.

Results showed that α-santalol at concentration of 25 to 75 μM resulted in a concentration-and time-dependent decrease in cell viability from 24 h treatment in both A431 and UACC-62 cells. Cell cycle analysis by flow cytometry showed that α-santalol at 50 μM and 75 μM induced G2/M phase cell cycle arrest starting from 6 hour treatment in both UACC-62 and A431 cells. Furthermore, Western immunoblotting data showed that α-santalol altered the expressions of cell cycle proteins such as cyclin A, cyclin B1, cell division cycle 2 (cdc2), cdc25c and cyclin-dependent kinase 2 (Cdk2) in A431 cells which are required for G2/M transition. α-Santalol treatment upregulated the expression of p21CIP1/WAF1 and suppressed the expression of mutated p53 in A431 cells; whereas, α-santalol treatment increased the expressions of wide-type p53 in UACC-62 cells.

Taken together, this study for the first time identifies the effects of α-santalol in G2/M phase arrest and describes the detailed mechanisms of G2/M phase arrest by this agent in both p53 mutated A431 cells and p53 wide-type UACC-62 cells, which might be contributing to its overall cancer preventive efficacy in various mouse skin cancer models in addition to induction of apoptosis by α-santalol. (Supported by 2010 Research Initiative Grant from the State of South Dakota funded as Translational Cancer Research Center).

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1083.
Abstract 1107: Mutant p53 increases epithelial mesenchymal transition, estrogen receptor alpha and erbB2/Her2/neu
Authors: Strasberg- Rieber Mary , Rieber Manuel
Published: February 2011

OBJECTIVES.- Progression of carcinomas involves transition from an epithelial cobblestone-like cytoarchitecture to an elongated, mesenchymal phenotype., leading to an epithelial to mesenchymal transdifferentiation (EMT) (1). Since the p53 tumor suppressor pathway is inactivated in most human cancers due to gene mutations or dysfunction, we now investigated in human wt p53 breast carcinoma cells, whether EMT and cell proliferation are influenced by introducing a dominant-negative (DN) R175H mutant p53 (2). This mutation occurring in the p53 zinc-binding domain, is common to breast cancer patients poorly responsive to therapy (2) METHODS.- .- Fluorescence microscopy was used to determine EMT-linked decreases in E-cadherin and cytokeratin 18. These changes and others associated with EMT, were verified by immune blotting. Laser scanning cytometry was used to measure expression of ErbB2 and estrogen receptor alpha, cell cycle profile and proliferation (2). RESULTS.- Cells transduced with the DN-R175H mutant p53, increased expression of the mesenchymal marker vimentin and proliferation-associated cyclin A, also showing a greater G2 population which correlated with increased c-neu/Her2/ErbB2. Joint cytometric analysis of the Ki67proliferation marker and estrogen receptor alpha revealed a greater number of estrogen receptor-positive proliferating cells (3) among the mesenchymal population harbouring the DN-R175H mutant p53 .CONCLUSIONS.- These results suggest that EMT induced by the DN-R175H mutant p53 promotes a gain of pro-oncogenic function by increasing the % of proliferating cells expressing estrogen receptor alpha (3) and the expression of erbB2 (4)

REFERENCES

1.-Kalluri R, Weinberg RA. The basics of epithelial-mesenchymal transition. J Clin Invest. 2009 119: 1420-8.

2.- Rieber M, Strasberg Rieber M. DN-R175H p53 mutation is more effective than p53 interference in inducing epithelial disorganization and activation of proliferation signals in human carcinoma cells : role of E-cadherin. Int J Cancer. 2009; 125:1604-12.

3.- Shoker BS, Jarvis C, Clarke RB, Anderson E, Hewlett J, Davies MP, Sibson DR, Sloane JP. Estrogen receptor-positive proliferating cells in the normal and precancerous breast. Am J Pathol 1999, 155:1811-1815.

4.- Cicalese A, Bonizzi G, Pasi CE, Faretta M, Ronzoni S, Giulini B, Brisken C, Minucci S, Di Fiore PP, Pelicci PG. The tumor suppressor p53 regulates polarity of self-renewing divisions in mammary stem cells. Cell. 2009; 138:1083-95.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1107.
Abstract 1130: ING4 tumor suppressor binds to a transcription co-activator, CCAR1, and modulates estrogen-dependent cell growth and gene expression
Authors: Dempsey Katherine , Whitsett Timothy G. , Kim Suwon
Published: February 2011

The tumor suppressor ING4 (Inhibitor of Growth family member 4) has been shown to play a role in diverse cancer-related cellular processes including gene regulation, apoptosis, cell migration, contact inhibition, and angiogenesis. Deletion or reduced expression of ING4 has been shown in various human malignancies including breast cancer, suggesting the tumor suppressive role of ING4. ING4 has been shown to interact with a number of proteins in different cell types, that include p53, NFkB, p300, HBO1-HAT complex, and HPH1. However, the binding partner(s) relevant to the ING4 tumor suppressor function in breast cancer have not been identified. We employed a bacterial two-hybrid screening system using the N-terminal half of the ING4 protein as bait and identified a number of candidate ING4 binding proteins (IBPs). One of the candidate IBPs was CCAR1 (Cell-Cycle and Apoptosis Regulator Protein 1). CCAR1 has recently been characterized as a transcriptional coactivator that is required for estrogen-induced gene expression and cell growth in human breast cancer cells (Kim JH, Yang CK, Heo K, Roeder RG, An W, and Stallcup MR, Mol Cell, 2008). We show the full length ING4 co-immunoprecipitated with endogenous CCAR1 in MCF7 breast cancer cells. In addition, we show that overexpression of ING4 blocked estrogen-dependent cell proliferation of MCF7 cells. These results indicated that ING4 directly binds to CCAR1 and may antagonize the CCAR1 activity required for estrogen-dependent cell growth. We conclude that ING4 may function as a tumor suppressor in breast cancer by modulating estrogen/CCAR1-dependent cell proliferation and gene transcription.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1130.