bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3788: Critical role of adapter protein p66shc in phenethyl isothiocyanate-induced apoptosis in human prostate cancer cells
Authors: Xiao Dong , Singh Shivendra V.
Published: February 2011

Phenethyl isothiocyanate (PEITC), a cancer chemopreventive constituent of edible cruciferous vegetables such as watercress, suppresses growth of human prostate cancer cells by causing apoptosis but the mechanism of cell death induction is not fully understood. Here, we provide experimental evidence to implicate adapter protein p66shc in regulation of PEITC-induced apoptotic cell death. Mouse embryonic fibroblasts lacking p66shc isoform were resistant to PEITC-mediated growth inhibition, cytoplasmic histone-associated apoptotic DNA fragmentation, and caspase-3 activation compared with wild-type fibroblasts. The PEITC treatment resulted in induction as well as increased Ser36 phosphorylation of p66shc in PC-3 (an androgen-independent cell line lacking functional p53) and LNCaP (an androgen-responsive cell line with intact p53) human prostate cancer cells. Moreover, siRNA knockdown of p66shc protein in PC-3 and LNCaP cells conferred significant protection against PEITC-induced cytoplasmic histone-associated DNA fragmentation, caspase-3 activation, and production of reactive oxygen species. Immunoprecipitation-immunoblotting experiments revealed increased binding of p66shc with prolyl isomerase 1 (Pin1), which is implicated in translocation of p66shc to mitochondria, upon treatment of PC-3 and LNCaP cells with PEITC compared with vehicle-treated control cells. Consistent with these results, treatment of PC-3 cells with PEITC resulted in translocation of p66shc to the mitochondria as judged by immunoblotting using cytosolic and mitochondrial fractions and immunofluorescence microscopy. Growth of PC-3 cells subcutaneously implanted in male athymic mice was significantly retarded by gavage with 9 μmol PEITC five times per week. The PEITC-mediated inhibition of PC-3 xenograft growth correlated with reduced cell proliferation, increased apoptosis, hyperphosphorylation of p66shc, and down-regulation of Pin1 protein level. Collectively, these results provide novel insight into the mechanism of PEITC-induced apoptosis to indicate critical role of p66shc in regulation of cell death. This investigation was supported by the National Cancer Institute grant 2 RO1 CA101753-06.

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 3788.
Abstract 3792: Phenethyl isothiocyanate inhibits oxidative phosphorylation to initiate reactive oxygen species-mediated death of human prostate cancer cells
Authors: Powolny Anna A. , Xiao Dong , de Moura Michelle Barbi , Bommareddy Ajay , Hahm Eun-Ryeong , Houten Bennett Van , Singh Shivendra V.
Published: February 2011

Phenethyl isothiocyanate (PEITC), a constituent of cruciferous vegetables (e.g., watercress), not only affords significant protection against chemically-induced cancer in experimental animals but also inhibits growth of human cancer cells in association with induction of apoptosis and autophagy. However, the mechanism by which PEITC causes cell death is not fully understood. Using LNCaP and PC-3 human prostate cancer cells as a model, we now demonstrate that the PEITC-induced cell death is initiated by reactive oxygen species (ROS) production due to inhibition of oxidative phosphorylation. Exposure of prostate cancer cells to pharmacologic concentrations of PEITC resulted in ROS production, which was accompanied by inhibition of complex-III activity, suppression of oxidative phosphorylation, and ATP depletion. These effects were not observed in a normal human prostate epithelial cell line (PrEC). The Rho-0 variants of LNCaP and PC-3 cells lacking normal oxidative phosphorylation were significantly more resistant to PEITC-mediated ROS generation, apoptotic DNA fragmentation, caspase-3 activation, and collapse of mitochondrial membrane potential compared with respective wild-type cells. The PEITC treatment resulted in activation of Bax in wild-type LNCaP and PC-3 cells, but not in their respective Rho-0 variants. Furthermore, Bax and Bak knockdown conferred significant protection against PEITC-induced apoptosis. The Rho-0 variants of LNCaP and PC-3 cells also resisted autophagy induction by PEITC treatment. In conclusion, the present study provides concrete experimental evidence to implicate ROS in initiation of PEITC-induced apoptosis and autophagy in human prostate cancer cells. This investigation was supported by the National Cancer Institute grant 2 RO1 CA101753-06.

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 3792.
Abstract 3836: Regulation of class III PI3K by class I PI3K/Akt inhibits autophagy through phosphorylation of UVRAG
Authors: Shu Shaokun , Sun Mei , Kim Donghwa , Cheng Jin Q.
Published: February 2011

It has been well documented that the class I PI3K (PI3KC1)/Akt pathway inhibits whereas class III PI3K (PI3KC3) induces autophagy. UVRAG forms a complex with Beclin1 and PI3KC3 leading to activation of autophagy process. In this study, we demonstrate that PI3KC1/Akt inhibits PI3KC3 kinase activity and autophagy induced by different stimuli, including mTOR inhibitor rapamycin, suggesting PI3KC1/Akt regulation of autophagy independent of mTOR. Further, Akt directly phosphorylates UVRAG on serine-509, which results in disruption of UVRAG-Beclin1-PI3KC3 complex, and inhibition of PI3KC3 and autophagy. Inhibition of PI3KC1 or depletion of Akt increases the ability of UVRAG to bind to Beclin 1. UVRAG-S509A, which is not phosphorylated by Akt, enhances whereas UVRAG-S509D, which mimics the phosphorylation by Akt, reduces the interaction between UVRAG and Beclin1. Notably, UVRAG-S509A abrogates the effect of Akt on PI3KC3 and autophagy. These findings establish the cross-talk between class I and class III PI3K and indicate that PI3KC1/Akt regulates autophagy through inhibition of PI3KC3 by phosphorylation of UVRAG.

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 3836.
Abstract 388: Epithelial to mesenchymal transition is associated with acquired resistance to sunitinib in patients with renal cell carcinoma: evidence from a xenograft study
Authors: Hammers Hans J. , Verheul Henk , Salumbides Brenda , Sharma Rajni , Jaspers Janneke , Rudek Michelle , Netto George , Pili Roberto
Published: February 2011

Purpose: Tyrosine kinase inhibitors (TKIs) targeting angiogenesis via inhibition of the vascular endothelial growth factor (VEGF) pathway have changed the medical management of metastatic renal cell carcinoma. While the treatment with TKIs has demonstrated clinical benefit these drugs will eventually fail patients. The potential mechanisms of resistance to TKIs are poorly understood. Experimental Design: To address this question we obtained an excisional biopsy of a skin metastasis from a patient with clear cell renal carcinoma who initially had a response on sunitinib and eventually progressed on therapy. Tumor pieces were grafted subcutaneously in athymic nude mice. Established xenografts were treated with sunitinib. Tumor size, microvascular density and pericyte coverage were determined. Plasma as well as tissue levels for sunitinib were assessed. A tumor derived cell line was established and assessed in vitro for a potential direct antitumor effects of sunitinib. Results: To our surprise, xenografts from the patient who progressed on sunitinib regained sensitivity to the drug. At a dose of 40 mg/kg sunitinib caused regression of the subcutaneous tumors. Histology showed a marked reduction in microvascular density and pericyte dysfunction. More interestingly, histological examination of the original skin metastasis revealed evidence of epithelial-to-mesenchymal-transition while the xenografts showed reversion to the clear cell phenotype. In vitro studies showed no inhibitory effect on tumor cell growth at pharmacologically relevant concentrations. Conclusions: The histological examination in this xenograft study suggests that reversible epithelial-to mesenchymal-transition may be associated with acquired tumor resistance to TKIs in patients with clear cell renal carcinoma.

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 388.
Abstract 3892: Chromosome instability mediated by impaired AuroraB kinase activity on kinetochores in CIN cancer cells
Authors: Haomin Huang , Yen Timothy J.
Published: February 2011

Aneuploidy and chromosome instability (CIN) are hallmarks of solid tumors. Recent studies showed that chromosome missegregation caused by defective kinetochore microtubule attachments during division can give rise to the CIN phenotype. However, the underlying molecular defect that is responsible for the CIN phenotype is poorly understood. On the other hand, increasing evidence suggests that aneuploidy is detrimental to cell growth. Similarly, how CIN cancer cells tolerate a continual state of aneuploidy also remains to be clarified. These are critical questions, given that CIN provides tumor cells a rapid way to adapt to stressful conditions such as chemotherapy.

In this study, we examined a panel of ovarian tumor cell lines and report that they exhibit the CIN phenotype. Timelapse movies showed chromosomes in these cell lines were able to align at the spindle equator but exhibited high rates (numbers) of lagging chromosomes, indicative of aberrant kinetochore: microtubule attachments. This observation was confirmed by the finding that a large fraction (numbers) of kinetochores in these cell lines exhibited a variety of attachment defects relative to Hela cells. As the role of Aurora B kinase at kinetochores is to monitor and repair defective microtubule attachments, we quantitated the levels of Aurora B at kinetochores of these ovarian tumor cell lines. We report that Aurora B levels are reduced relative to Hela cells that do not exhibit attachment defects. Consistent with this finding, FRET studies to monitor the real-time Aurora B kinase activity at the kinetochores of two of the three ovarian cell lines showed that kinase activity was weak. This defect was localized to kinetochores, as the overall activity of Aurora B kinase, as monitored by the western blotting of phospho-Histone H3, was similar to that in Hela cells. These findings provide a molecular explanation for kinetochore attachment defects in these ovarian tumor cell lines and, as a consequence, their chromosome instability.

We also analyzed the response of these cell lines to treatment with various anti-mitotic agents. We found no obvious growth advantage of these ovarian CIN cell lines in response to drugs, relative to Hela cells. Sensitivity to mitotic death may reflect the variation of the genetic background of a given cell line. Also, the mitotic checkpoint appeared to be required for death in mitosis. Although apoptosis clearly played an important role in mitotic cell death in our studies, other death pathways could participate in this event, when apoptosis was blocked.

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 3892.
Abstract 390: Correlation of the effects of antiangiogenic agents aflibercept (VEGF Trap) and anti-Dll4 antibody on tumor vascular perfusion and tumor growth
Authors: Adler Alexander P. , Eichten Alexandra , Parveen Asma A. , Thurston Gavin
Published: February 2011

Anti-angiogenic treatments are becoming widely used in several major cancer types. Most experimental and clinical development to date has focused on the VEGF pathway, although other signaling pathways, including Dll4-Notch, are also now being explored. However, clinical development for anti-angiogenic therapy has been hindered by a lack of tumor response biomarkers. In this study, we sought to correlate long-term tumor growth responses following blockade of VEGF or Dll4 with rapid effects on tumor vascular perfusion. Using VEGF Trap (aflibercept) and a blocking Dll4 antibody, we compared two tumors that differ widely in their long-term growth responses to each agent: C6 rat glioma tumors (anti-Dll4 resistant, aflibercept sensitive) and HT1080 human fibrosarcoma (anti-Dll4 sensitive, aflibercept resistant), both grown subcutaneously in SCID/CB17 mice. Tumor vascular perfusion was assessed using contrast-enhanced micro-ultrasound at 24 hr after treatment with aflibercept, anti-Dll4 Ab, or the combination, and tumor growth was measured during the course of prolonged twice-weekly treatments. We found that aflibercept treatment rapidly decreased tumor vascular perfusion in C6 tumors (36%) and produced an extended delay in tumor growth (8 days to reach 500mm3). In contrast, anti-Dll4 antibody produced no significant decrease in perfusion of C6 tumors at 24 hr after treatment, and a less dramatic delay in tumor growth (5 days to reach 500mm3). In HT1080 tumors, anti-Dll4 Ab dramatically decreased perfusion (38%), as well as causing a prolonged growth delay (20 days to reach 300mm3), whereas aflibercept caused neither a decrease in perfusion nor growth in these tumors. Unexpectedly, combination treatment with aflibercept and anti-Dll4 was most potent in both tumor types at reducing tumor perfusion and inhibiting tumor growth. Analysis of vascular density by CD31 immunohistochemistry at 24 hr after treatment revealed a decrease in C6 (57% and 31%) and HT1080 (28% and 19%) upon single agent aflibercept and combination treatment, respectively, while anti-Dll4 increased apparent vascular density of C6 tumors (31%) but produced no change in HT1080 tumors. Taken together, these data show that changes in tumor perfusion, but not in vessel density, correlate with long-term tumor responses to aflibercept and anti-Dll4, thus suggesting that decreases in tumor perfusion could be a rapid response biomarker for efficacy of anti-angiogenic therapies.

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 390.
Abstract 3905: Structure–function relationships between RUNX1-MTG fusion proteins and core components of the Notch transcription complex
Authors: Mariotti Jolene , Nguyen HN , Engel Michael
Published: February 2011

The myeloid translocation genes (MTG) are preferred targets of chromosomal translocations with the Runx1 gene in acute myeloid leukemia (AML) pathogenesis. The resulting fusion proteins, RUNX1-MTG8 and RUNX1-MTG16, influence gene expression programs that control self-renewal and differentiation in hematopoiesis. How they engage the machinery of self-renewal to alter hematopoietic development is poorly understood. To gain additional insights into this question, we investigated structure–function relationships between RUNX1-MTG fusion proteins and core components of the Notch transcription complex, a critical determinant of cell fate specification in diverse tissues. The Notch transcription complex is composed minimally of the transcriptional repressor CSL, the intracellular domain of Notch receptors (N-ICD) and the transcriptional co-activator Mastermind (MAML). We show that RUNX1-MTG fusion proteins transactivate a Notch-responsive element from the Hes1 promoter. Using MTG16 as a model protein, we show that N-ICDs interact with a discrete domain in the MTG N-terminus. We translated these findings to the fusion proteins and found N-ICD binding was retained by RUNX1-MTGs and a RUNX1-MTG16 mutant that lacks the N1-ICD binding site. In parallel experiments, we defined the binding region for MTG proteins and their fusion protein derivatives on the N1-ICD. Informed by these structural relationships, we utilized RUNX1-MTG proteins or mutants that lack N1-ICD binding sites in transcriptional reporter assays. Our findings suggest that RUNX1-MTG fusion proteins alter transcription of the Notch target gene, Hes1 and intimate a structure–function relationship between RUNX1-MTG fusion proteins and Notch signaling.

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 3905.
Abstract 3906: Twist regulates estrogen receptor expression in breast cancer
Authors: Vesuna Farhad , Lisok Ala , Kimble Brian , Domek John , Kato Yoshinori , Artemov Dmitri , Kowalski Jeanne , Carraway Hetty , van Diest Paul , Raman Venu
Published: February 2011

Background: Estrogen receptor alpha (ER) plays a crucial role in breast cancer development and progression due to its role as an effector/mediator of the mitogenic hormone estrogen. However, the role of transcriptional regulation of ER in tumor progression is not completely characterized. While defining the role of the basic helix loop helix transcription factor Twist (TWIST1) in breast cancer development, we identified ER as a downstream target of Twist in breast cells.

Methods: A knock-up and knock-down approach in Twist negative and positive cell lines was used in order to characterize the regulation of ER by Twist. We analyzed ER expression by immunoblotting, ER transcriptional activity by promoter assays, DNA binding by chromatin immunoprecipitation, and estrogen independent growth in the presence of stripped serum or the estrogen antagonist tamoxifen (TAM). Additionally, ER promoter methylation was measured by methylation specific quantitative PCR (MS-qPCR). Using orthotopic xenografts in SCID mice, tumor growth was determined in presence of TAM and assayed for vascular permeability and volume by functional magnetic resonance imaging. Finally, the relationship between Twist and ER expression in breast tumors was evaluated by quantitative reverse-transcription PCR (qRTPCR).

Results: Twist transcriptionally down-regulated ER expression by direct promoter modulation. Twist over-expression caused estrogen independence and TAM resistance, and induced hypermethylation of the ER promoter. In vivo, Twist expression resulted in estrogen independent growth and resistance to TAM. Twist over-expressing tumor xenografts exhibited high vascular volume and permeability. Moreover, Twist and ER expression were inversely correlated in primary breast cancer patient samples. Conclusion: The regulation of ER by Twist might be an underlying mechanism for ER negativity seen in breast tumors and could have important clinical implications for ER negative breast cancers.

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 3906.
Abstract 3917: Molecular mechanisms underlying hedgehog-GLI oncogenic function via interaction with the TGF-beta-SMAD4 axis
Authors: Nye Monica D. , Cross Aishah , Almada Lucianna L. , Elsawa Sherine F. , Fernandez-Zapico Martin E.
Published: February 2011

Crosstalk between signaling pathways is crucial to the initiation, progression and maintenance of the transformed phenotype. Therefore, characterization of these signaling interactions is fundamental for the understanding of the complex and varied network of events that leads to the development and spread of tumors. The Hedgehog (HH)-GLI pathway, an important carcinogenic cascade, is required for the growth and survival of tumors of diverse origin. Recent reports, mainly from animal models, suggest that key signaling interactions are required by HH-GLI signaling to modulate cancer-associated cellular functions. However, the molecular mechanisms underlying this phenomenon in cancer and non-cancer cells remain elusive. Our preliminary data show a crosstalk between the HH-GLI axis and TGF-beta pathway, a well-characterized tumor suppressor/oncogenic signaling pathway. Luciferase reporter and expression assays in combination with genetic and pharmacological manipulations of the TGFbeta pathway show that this cascade is able to activate GLI transcriptional activity, even in the absence of an active HH signaling. The TGFbeta pathway is required to maintain active GLI transcriptional complexes at GLI target genes. Further analysis of this molecular interaction suggests that TGFbeta pathway-mediated activation of GLI transcription factors requires an intact SMAD4, thus suggesting a potential mechanism mediating this functional interaction. The knowledge derived from this study provides both mechanistic insight into the contribution of this HH-GLI/TGF-beta pathway interaction to carcinogenesis and could serve as a foundation for development of new therapeutic approaches for tumors with active HH-GLI and TGF cascades.

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 3917.
Abstract 3919: Potential roles of oncogenic ΔNP63α during bone and bone cancer formation
Authors: Abbassi Sam , Ding Ming , Li Feifei , Lu Yaojuan , Wu Guojun , Zheng Qiping
Published: February 2011

P63 belongs to the P53 tumor suppressor gene family. Due to its alternative promoter usage and splicing, p63 consists of two major subtypes (TAp63 and ΔNp63) and six different isoforms (TAp63α, β, or γ and ΔNp63α, β, or γ). These isoforms have been shown to play distinct biological functions in cancer and development. P63, especially the ΔNp63α variant, has been suggested to play an oncogenic role in cancer progression, including bone-related tumors such as giant cell tumor of bone and osteosarcoma. Interestingly, P63 has previously been shown to be an essential player for limb development. This raises the possibility that there might be a specific mechanism linking P63 regulation of bone and bone cancer formation.

The type X collagen gene (Col10a1) is a specific marker of hypertrophic chondrocytes during endochondral bone formation. As a matrix protein, type X collagen also functions as a regulatory molecule that affects chondrocyte mineralization, apoptosis, and angiogenesis. Therefore, transcriptional regulators critical for Col10a1 expression are also expected to affect the apoptotic pathway that occurs during bone cancer formation. By real-time RT-PCR and proteomic approaches using a MCT cell model, we demonstrate here that P63 contributes to regulation of Col10a1 expression in vitro. We also have data which supports that Runx2 regulates cell-specific Col10a1 expression via the Runx2 binding sites found within the Col10a1 cis-enhancer. This is intriguing since p63 was previously shown to interact with Runx2 by yeast two-hybrid approach. Moreover, we have generated a mouse model in which Runx2 was driven by the cell-specific Col10a1 control element. Our preliminary results showed that these Col10a1-Runx2 transgenic mice have enhanced Runx2 and Col10a1 expression, a longer hypertrophic zone, altered chondrocyte maturation, apoptosis, and disturbed ossification. These results suggest the decreased apoptotic activity mediated by Runx2, whereas elevated Runx2 expression has been associated with osteosarcoma development and chondrosarcoma progression. To address the potential in vivo relevance of P63 on bone and cancer formation, we have established Col10a1-ΔNp63α transgenic mouse lines using the same Col10a1 control element. Characterization of these transgenic mice will allow us to study if this oncogenic P63 isoform regulates Col10a1 expression in vivo and if this regulation is within the network regulating both bone and bone cancer formation.

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 3919.
Abstract 3961: Chemotherapy treatment induces an Interleukin-6 deficit in the bone marrow microenvironment
Authors: Rellick Stephanie , Piktel Debra , Walton Cheryl , O'Leary Heather , Othumpangat Sreekumar , Hall Brett , Craig Michael , Gibson Laura F.
Published: February 2011

Bone marrow stromal cells (BMSC) are a critical component of the microenvironment that supports hematopoiesis, and hematopoietic recovery following bone marrow transplantation. Aggressive chemotherapy not only affects tumor cells, but also influences structural and functional components of the microenvironment, including BMSC and osteoblasts. Successful stem cell or bone marrow transplantation following immuno-suppressive or myeloablative chemotherapy is dependent on the ability of BMSC, and other cellular components of the microenvironment, to maintain their functionality. This includes secretion of soluble factors and expression of cellular adhesion molecules that are critical for the survival, proliferation, and differentiation of immature progenitor cells. In the current study, we have investigated the effects of chemotherapy treatment on BMSC and human osteoblast (HOB) expression of Interleukin-6 (IL-6). IL-6 is a pleiotrophic cytokine which has diverse effects on hematopoietic cell development. The treatment of BMSC or HOB with Melphalan or VP-16, which are clinically relevant drugs used in pre-transplant regimens, led to decreased IL-6 protein expression. This decrease in IL-6 protein is unique to Melphalan, as treatment with other chemotherapeutic agents does not result in a similar decrease in IL-6 protein. We have also observed a decrease in gp130, the transmembrane protein necessary for IL-6 signaling, in response to Melphalan treatment, but not VP-16 treatment. Additionally, pre-treatment of BMSC and HOB with Z-VAD-FMK or Leupeptin, a broad range caspase inhibitor and lysosomal inhibitor respectively, prior to chemotherapy treatment was not able to blunt the decrease in IL-6 protein detected in cell supernatants. These data suggest chemotherapy is not enhancing the degradation of IL-6 protein through caspase or lysosomal mediated mechanisms, but rather blunting the overall expression of IL-6. Melphalan treatment is also able to decrease IL-6 mRNA and further investigation is necessary to determine the mechanism by which chemotherapy affects transcription of IL-6. Collectively, these observations suggest that chemotherapy treatment induced alteration of bone marrow microenvironment function, which results in decreased or defective hematopoietic support of human embryonic stem cells and early progenitor cells may result, in part, from an IL-6 deficit.

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 3961.
Abstract 3974: TIAF1 blocks p53 suppression of SMAD promoter activation
Authors: Chang Nan-Shan , Hsu Li-Jin , Lee Ming-Hui , Lin Sing-Ru
Published: February 2011

p53 participates in the TGF-β signaling. How p53 regulates SMAD gene transcription is largely unknown. TIAF1 is a TGF-β1-induced factor and a downstream effector of the TGF-β signaling. TIAF1 is known to control nuclear localization of Ser15-phosphorylated p53. Ectopic TIAF1 induces growth inhibition and apoptosis of monocytic U937 and many non-fibroblasts, via upregulation of p53, p21, and Smad2/4, but downregulation of ERK phosphorylation. Wild type p53 suppresses the transcriptional activation of SMAD gene, whereas isoform p53γ induces the gene activation. TGF-β1 increases the physical association of TIAF1 with Smad4, and when overexpressed, TIAF1 inhibits Smad4-dependent promoter activation. In combination, TIAF1 and p53 support the promoter activation regulated by endogenous or ectopic Smad4, indicating that TIAF1 blocks p53 suppression of the SMAD promoter activation, and vice versa. Without Ser46 phosphorylation in p53 (p53ΔS46), p53ΔS46 alone, or in the co-presence of TIAF1, abolishes the Smad4-regulated promoter activation. These observations suggest a critical role of Ser46 in p53 in controlling SMAD transcriptional activation.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

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 3974.
Abstract 3980: Quantitative detection of discriminatory proteins in tissue and serum of ovarian cancer patients by LC-MSE
Authors: Wegdam Wouter , Kramer Gertjan , Langridge James , Moerland Perry D. , de Jong Shreyas M. , Vissers Johannes P. , Kenter Gemma G. , Buist Marrije R. , Aerts Johannes M.F.G.
Published: February 2011

Purpose

To search for potential protein markers or activated pathways which play a role in ovarian cancer.

Experimental procedures

Serum of six patients with a serous adenocarcinoma of the ovary (stage IIIB or higher) was collected before treatment. Matched control patients with a serous cystadenoma of the ovary were chosen as a control group. In addition, homogeneous regions of cells that exhibited uniform histology were isolated from cancer tissue in three of these patients by laser capture microdissection (LCM), normal ovarian epithelial cell samples were taken from their matched controls. Subsequently we employed label-free liquid chromatography tandem mass spectrometry (LC-MSE) to identify proteins and determine their absolute concentrations in serum specimens and tissue lysates. Moderated t-tests were used to identify differentially expressed proteins between patient- and control-group, p-values were adjusted for multiple-testing using Benjamini-Hochberg false discovery rates. Proteins were considered to be differentially expressed if the adjusted p-values were <0.05.

Summary of the data

In the serum specimens, 13 differentially expressed proteins were identified by LC-MSE profiling. Proteins with different concentrations in patients versus control sera include abundant serum proteins such as apolipoprotein AI and transferrin, that both exhibited a lower concentration in serum of cancer patients. Differential expression was also observed for apolipoproteins (APOA IV, APOA II and APOC III) and other proteins that have not been associated with ovarian cancer previously such as C9 and Afamin.

In the tissue lysates, 17 differentially expressed proteins were identified. Amounts of collagen found were decreased in cancer tissue. Furthermore, proteins associated with the respiratory chain and cell migration were found in increased amounts in the cancer tissue lysates.

Interestingly, no overlap was found between the discriminatory proteins in serum specimens and corresponding tissue lysates, illustrating the challenge to identify a single biomarker in the circulation reflecting ovary cancer.

Conclusions

Our study revealed several protein changes in serum and the ovary of patients suffering from serous adenocarcinoma of the ovary (stage IIIB or higher). Further investigation of these proteins is warranted to establish whether they could provide new insights into the etiology of the disease and act as potential new disease markers.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

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 3980.
Abstract 3998: Development of angiogenesis and cancer cell invasion assays in microfluidic flow cells for use with real-time microscopy
Authors: Conant Carolyn G. , Schwartz Michael , Ionescu- Zanetti Cristian
Published: February 2011

Understanding the molecular and cellular mechanisms of cancer progression from primary malignancy to metastatic disease is critical to development of successful treatments. Cell invasion from the primary tumor and tumor-induced angiogenesis are just two of many phenomena contributing to the pathology of metastatic disease. Both biological processes involve migration and transmigration of cells in response to chemoattractants. In vitro analysis of these phenomena typically involves deposition of a basement membrane derived matrix (Matrigel) in a transwell plate insert. This allows quantitation of either invasion or angiogenesis, but the inserts can be cumbersome to process. Here, we demonstrate a microfluidic based method to follow angiogenesis or cell invasion in real time. The method enables acquisition of high content data by microscopy using microfluidic flow cells which are optimized for imaging. The channels were filled with matrix in one half of the channel while the other half was maintained in the fluid phase. Using this technique, we tested endothelial cell response to VEGF and fumagilin. It was shown that VEGF impregnated in the matrix promoted angiogenesis while fumagilin abrogated the effect. We also investigated invasion of serum starved HT1080 and MCF-7 cells into FBS containing matrix and found that as predicted only HT1080 cells successfully invaded the matrix. Using these experimental conditions, the method is amenable to screening many cell-types or many simultaneous conditions or compounds by microscopy in the solid or liquid matrix of a microfluidic channel.

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 3998.
Abstract 4021: Genetic studies identify a map of redox signaling pathways in Drosophila
Authors: James Brian P. , Staatz William , Clark Elizabeth , Lu Jonathan , Powis Garth
Published: February 2011

Altered redox signaling, a consequence of oxidative stress and hypoxia, is seen in many human cancers and there is compelling evidence that altered redox signaling leads to tumorigenesis and aggressive tumor growth. In order to survive redox stress, tumor cells must initiate an adaptive response that facilitates both survival and drug resistance. The response to oxidative stress is largely restricted to tumor cells and thus offers a selective target for drug development. While it is clear that redox signaling is an important cellular response, our understanding of redox signaling pathways is rudimentary, and a map of redox signaling pathways comparable to that for protein phosphorylation is not available. In order to identify novel redox signaling genes we have undertook a classical dominant modifier screen in Drosophila melanogaster. Redox homeostasis is maintained in humans and Drosophila by scavenger systems such as superoxide dismutase (SOD), glutathione peroxidase and thioredoxin (Trx). In humans, Trx and glutathione are reduced and regenerated by thioredoxin reductase (Trxr) and glutathione reductase. Drosophila, and possibly insects in general, have only one glutathione/thioredoxin reductase gene encoded by the thioredoxin reductase-1 gene (Trxr-1). Thus, in Drosophila Trxr-1 serves as the starting point for all redox reactions in the cell and mutational disruption of the Trxr-1 gene leads to a genetically redox stressed animal suitable for screening. We screened a collection of second and third chromosome deficiency lines to identify genomic regions that dominantly suppress, or enhance the semi-lethal phenotype of Trxr-1 mutants. Our initial screen identified 18 genomic regions that dominantly suppress or enhance Trxr-1 mutants. We used overlapping deficiencies to validate and narrow down 9 genomic regions and have identified six individual genes with multiple alleles that show suppressing or enhancing phenotypes in four of these regions including mutations in two genes associated with lipid signaling and the Toll-like receptor, 18-Wheeler. The identification of 18-Wheeler indicates that the screen was successful, as 18-Wheeler, in addition to playing a critical role in the Drosophila immune response, is a member of the redox-regulated NF-κB pathway. This screen identified several genes not previously known to be involved in redox signaling, and begins to define a map of redox signaling pathways likely conserved through evolution.

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 4021.
Abstract 4067: A T-cell factor-4 motif regulates the phenotype of hepatocellular carcinoma cells
Authors: Koga Hironori , Walker Evan J. , Tsedensodnom Orkhontuya , Wands Jack R. , Kim Miran
Published: February 2011

T-cell factor (TCF) family proteins play a crucial role in regulating the activation of Wnt/β-catenin responsive target genes. Among the family proteins, TCF-4 is predominantly expressed in human HCC tissues. We have recently cloned 14 TCF-4 isoforms from human hepatoma cell lines, and naming them in alphabetical order from A to M. A TCF-4J and K pair has been identified based on the presence (K) or absence (J) of the SxxSS motif at the beginning of exon 9. This motif may modulate transcriptional activity of TCF-4 through phosphorylation/dephosphorylation of serine residues (Pukrop et al., Cancer Res 2001). However, cell-based functional analysis of this motif has yet to be performed. The AIM of this study was to investigate whether TCF-4J and K have distinct cellular phenotypes due to SxxSS motif expression. Methods: The human hepatoma cell line Huh-7 was used in this study. The cells were transfected with TCF-4J and K expression plasmids, and protein levels in cell lysates and nuclear extracts were examined by Western blot analysis. TCF-4K mutants with substitution of serine (S) to alanine (A) were generated by site-directed mutagenesis. Results: Protein expression of all cloned TCF-4 isoforms was confirmed by Western blot analysis. Expression levels of cyclin D1, which is a direct target of TCF-4, was increased in both cell lysates and the nuclear extracts of the TCF-4K, but not in TCF-4J-transfected cells. The TCF-4K isoform with a S269A mutation revealed a decrease in the expression level of cyclin D1, while the mutant with either a S272A or S273A substitution did not, which suggests that phosphorylation of serine 269 was critical to up-regulating cyclin D1 gene expression. Conclusion: The findings from functional analysis following mutagenesis of the SxxSS motif suggest that serine phosphorylation of this motif resulted in the altered target genes expression, including cyclin D1. Therefore, TCF-4 isoforms regulate the phenotype of HCC 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 4067.
Abstract 4071: Prohibitin peptides induce apoptosis in cancer cells and inhibit angiogenesis
Authors: Rizwani Wasia
Published: February 2011

Prohibitin is a potential tumor suppressor protein that can repress E2F-mediated transcription and arrest cell proliferation. E2F1 binding domain of prohibitin is a coiled-coil structure and prohibitin functions through this domain to repress E2F1 transcription activity by recruiting HDAC1. The coiled- coil domain (175-217aa) by itself exhibited growth suppressive properties and showed a reduction in colony formation when transfected into breast cancer cells, T47D cells. It was shown that a synthetic peptide (42aa), designated as H1-6, corresponding to the coiled- coil domain induced apoptosis in four different human cell lines; MCF7, HAEC, BL18 and WI38 cells. Hence we decided to synthesize peptides of 14-mer (H1-2, H3-4, H5-6) and 7-mer (H2, H5) from the coiled coil domain of prohibitin and study their effect on cells. We found that each one of the prohibitin peptides (20μM) induced a marked increase in apoptosis in a panel of breast, lung and prostate cancer cell lines when compared to the control peptide (CP1). Interestingly, the peptides had no effect on breast epithelial cells, MCF10A. Treatment of MDA-MB-231 cells with 20μM of each prohibitin peptide individually reduced the invasive capability of these cells when compared to the control peptide. We also investigated their effect on angiogenesis in HUVEC cells and found that these peptides could significantly inhibit angiogenesis in vitro. In conclusion, these results suggest that these peptides could be of significant therapeutic value.

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 4071.
Abstract 4073: The PNA oligonucleotide is effective miRNA inhibitor that transfection reagents are not required
Authors: Oh Su Young , Ju Yeong Soon , Kim Seryun , Park Heekyung
Published: February 2011

The miRNAs are approximately ∼23 nucleotides non-coding RNAs. They regulate major processes such as development, apoptosis, cell proliferation, hematopoiesis, and patterning of the nervous system. MiRNA inhibition using antisense oligonucleotide is an effective tool for understanding miRNA function. The peptide nucleic acid (PNA) is an artificial oligonucleotide with a peptide backbone. PNAs have a stronger affinity and greater specificity than DNA or RNA and are resistant to nuclease, which is essential characteristic for a miRNA inhibitor that will be exposed to abundant serum and cellular nucleases. In here, we evaluated PNAs™ miRNA inhibitors in cultured cells including stem cell using luciferase assay and qPCR. FACS analysis was showed that PNAs™ miRNA inhibitors can penetrate to intra cells without transfection reagent. Also, miRNA inhibition effects are sustained until 15 days after treatment. PNAs™ miRNA inhibitor has a lower cytotoxicity than other nucleic acid-based miRNA inhibitors. PNAs™ miRNA inhibitors can be stored at room temperature for 20 weeks without losing the activity. These results demonstrate that PNAs are a powerful and effective tool to studies of miRNA mechanism.

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 4073.
Abstract 412: Isolation and analysis of metastasis-related genes in an oral cancer cells with autocrine stromal cell-derived factor −1/CXCR4 system
Authors: Uchida Daisuke , Onoue Tomitaro , Kuribayashi Nobuyuki , Tamatani Tetsuya , Nagai Hirokazu , Miyamoto Youji
Published: February 2011

We have previously demonstrated that distant metastasis might require the autocrine stromal cell-derived factor-1 (SDF-1; CXCL12)/CXCR4 system in cases of oral squamous cell carcinoma (SCC). In this study, we examined the expression of metastasis-related genes in B88-SDF-1 cells, which have an autocrine SDF-1/CXCR4 system and distant metastatic potentials in vivo. Microarray analysis revealed that 418 genes were upregulated in B88-SDF-1 cells. To confirm the specificity of the microarray analysis, 3 highly upregulated genes, metabotropic glutamate receptor 5 (mGluR5), plasticity related gene 1 (PRG1), and unknown gene were reanalyzed by RT-PCR. These 3 genes were upregulated in B88-SDF-1 cells in comparison with those in mock cells, but AMD3100, a specific antagonist of CXCR4, abrogated the upregulation of 2 (mGluR5 and PRG1) out of 3 genes in B88-SDF-1 cells. Thus, we analyzed the expression of metabotropic glutamate receptor 5 (mGluR5), one of the genes that were upregulated in B88-SDF-1 cells. A specific mGluR5 antagonist, MPEP did not affect the growth of B88-SDF-1 cells, but did inhibit the enhanced-migration of B88-SDF-1 cells. When the B88-SDF-1 cells were transplanted into the vein, numerous metastatic foci were detected. Under the present conditions, MPEP significantly inhibited the lung metastasis of the B88-SDF-1 cells, ameliorated body weight loss, and improved the survival rate of tumor-bearing nude mice. These results suggested that mGluR5 might be one of a possible metastasis-related gene induced by an autocrine SDF-1/CXCR4 system in oral SCC.

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 412.
Abstract 4192: Global gene expression profiling confirms the molecular fidelity of primary tumor-based orthotopic xenograft mouse models of medulloblastoma
Authors: Zhao Xiumei , Zhang Yujing , Baxter Patricia , Yu Litian , Voicu Horatiu , Gurusiddappa Sivashankarappa , Lau Ching C. , Leung Hon-chiu Eastwood , Li Xiao-Nan
Published: February 2011

Clinically relevant animal models are needed for understanding biology and testing novel therapies in medulloblastoma (MB), the most common malignant brain tumor that occurs in children. We have recently established a panel of orthotopic xenograft mouse models of MB through direct injection of fresh surgical specimens into the cerebella of Rag2/SCID mice. The xenograft tumors were previously shown to have replicated key histopathological phenotypes and invasive growth characteristics of the original patient tumors. The objective of the current study is to complete the molecular characterization of 11 MB models that have been serially passaged in vivo in mouse brains for at least three generations. Whole genome gene expression profiling was performed in duplicate with Illumina HumanWG6 v3 gene expression profiling beadchip that contains more than 48,000 probes. Our results showed that the xenograft tumors are molecularly accurate. When the nine passage I xenograft tumors were compared with their original patient tumors, high correlation coefficiency were observed (r2 >0.95 in five models, >0.9 in three models, and >0.85 in one model). Serial subtransplantation up to five times did not cause significant changes of gene expression pattern (r2>0.9 in eight models, >0.8 in three models). Using RNAs from 4 normal fetal brains, one child cerebellum, and 5 adult cerebella as control, we further showed that these xenograft models, although molecularly independent displaying distinct gene expression profiles, can be classified into five different subcategories. A selected set of the differentially expressed genes were validated using real-time RT-PCR and Immunohistochemistry. In conclusion, this study provided strong experimental evidence to demonstrate that primary tumor-based orthotopic xenograft mouse models of MBs are molecularly accurate, and our findings of genetic abnormalities should significantly facilitate the rationally designed preclinical drug screenings in the near future.

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 4192.