bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4026: Id4 acts as a tumor suppressor by regulating c-Myc and MDM2 in prostate cancer
Authors: Evans Ashley L. , Carey Jason WP , Glymph Shanora E. , Chaudhary Jaideep
Published: June 2012

PURPOSE: Id proteins (Id1, Id2, Id3, and Id4) function as dominant negative regulators of basic helix loop helix transcription factor family. They share the conserved HLH domain but lack the DNA binding basic domain. This domain configuration allows the Id proteins to dimerize with bHLH proteins. The Id-bHLH dimer therefore fails to bind the E box consensus sequence and activate transcription. In general Id1 and Id3 promote proliferation and inhibit differentiation and thus their expression is generally high in many cancers. The function of Id4 appears to be unique as compared to Id1-Id3. Recent studies have demonstrated that Id4 is epigenetically silenced in many cancers suggesting that Id4 may in fact act as a tumor suppressor. The present study was carried out to determine the molecular mechanism by which Id4 acts as a tumor suppressor in prostate cancer. DESIGN METHODS: Initial studies were carried out on DU145 cells in which Id4 is epigenetically silenced. Over-expression of Id4 decreased proliferation and promoted epithelial like morphology in DU145 cells. A proteomics based approach suggested that MDM2 is a potential downstream target of Id4. The upstream regulators and downstream targets of MDM2 were then evaluated to understand the mechanism and functional significance of Id4-MDM2 cross talk. RESULTS: A combination of expression and immuno-localization studies demonstrated that c-Myc and p14ARF, the upstream regulators of MDM2 expression are down-regulated in the presence of Id4. Id4 also significantly up-regulated the expression and nuclear localization of CDKNIs p21and p27. Unexpectedly, the expression of p53 a known target of MDM2 was decreased both at the transcript and protein level. CONCLUSIONS: Although MDM2 was discovered as a potential downstream target, our pathway analysis suggests that c-Myc could also be a downstream target of Id4. The de-repression of CDKNI appears to be c-Myc dependent but p53 independent primarily because in DU145 cells, p53 is mutated. Alternatively, decreased MDM2 could promote p21 stability resulting in increased nuclear localization and expression. Taken together our data support the role of Id4 as a potential tumor suppressor that is unique and opposite to the oncogenic properties of other HLH members Id1, Id2 and Id3.

Supported by: NIH/NCI R01CA128914 and NIH/NCRR/RCMI G12RR03062.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4026. doi:10.1158/1538-7445.AM2011-4026
Abstract 404: Proinflammatory cytokine IL-1β stimulates IL-8 synthesis in mast cells via a leukotriene B4 receptor 2-linked pathway, contributing to angiogenesis
Authors: Lee Jin-Wook , Kim Hyunju , Wei Jun-Dong , Seo Ji-Min , Park Soo Young , Park Geun-Soo , Lee A-Jin , Kim Jae-Hong
Published: June 2012

Recent studies have suggested that mast cells have critical roles in angiogenesis. However, the detailed mechanism by which mast cells contribute to angiogenesis is not yet clearly understood, especially in response to proinflammatory cytokines. In this study, we showed that the proinflammatory cytokine IL-1β induces the synthesis of IL-8, a potent angiogenic factor, in human mast cells via the leukotriene B4 receptor (BLT)2. We also characterized the BLT2 downstream signaling pathway and determined that BLT2-mediated IL-8 synthesis involves the upregulation of Nox1, a member of the NADPH oxidase family, Nox1-dependent reactive oxygen species generation and the subsequent activation of the redox-sensitive transcription factor NF-κB. For instance, knockdown of BLT2 and Nox1 with specific small interfering RNA, treatment with a specific BLT2 antagonist, LY255283, or treatment with a potential Nox inhibitor, diphenylene iodonium, suppressed IL-1β-induced IL-8 synthesis. We found that the conditioned media collected from IL-1β-treated human mast cell line HMC-1 had significantly enhanced angiogenic activity that could be dramatically attenuated by either small interfering RNA knockdown of BLT2 or treatment with neutralizing Ab to IL-8. Finally, the experiments were repeated using human primary cord blood-derived mast cells, and the results were clearly reproduced. Taken together, our results suggest that BLT2-Nox1-reactive oxygen species-dependent pathway plays a role in promoting the secretion of IL-8 from human mast cells in response to the proinflammatory cytokine IL-1β, thus contributing to angiogenesis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 404. doi:10.1158/1538-7445.AM2011-404
Abstract 4040: Goyazensolide, a sesquiterpene lactone from Piptocoma rufescens inhibits tumor development in hollow fiber
Authors: Shen Qi , Matthew Susan , Wittwer Jennifer A. , Ren Yulin , Kinghorn A. Douglas , Carcache de Blanco Esperanza J. , Swanson Steven M.
Published: June 2012

Goyazensolide (C19H20O7) is a rare sesquiterpene lactone isolated from Piptocoma rufescens Cass. (Asteraceae). Goyazensolide significantly inhibits NF-κB transcription factor and decreased the cell growth of HT-29 colon cancer cells in a dose-dependent manner. In the present study, we investigated the effects of goyazensolide in a hollow fiber assay using in vivo setting. We also examined the involvement of ROS-signaling components (ROS levels, NF-κB transcription factor, mitochondrial membrane potential) and demonstrated that the antiproliferative effect of cancer cells is partially mediated by induction of oxidative stress. It is our aim to further investigate the molecular mechanism through which goyazensolide exerts its cytotoxic effect in vitro and its antitumor effect in vivo. Our data therefore supports goyazensolide as an active anti-proliferative agent with great potential for further development.

Acknowledgment

This research was supported by Program Project Grant P01-CA125066 funded by the National Cancer Institute, NIH.We also wish to acknowledge the plant taxonomists who collected the plant material used in this investigation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4040. doi:10.1158/1538-7445.AM2011-4040
Abstract 424: Interference with alphaVbeta3 integrins via cyclic RGD peptides affects breast cancer cell behavior
Authors: Martel Alexandra , Poettler Marina , Kalinowska Waclawa , Valent Peter , Zielinski Christoph , Prager Gerald W.
Published: June 2012

Integrin alphavbeta3 is mainly expressed in stimulated endothelial cells, thus it has recently become the focus of intense research mainly to interfere with tumorangiogenesis. However, alphavbeta3 integrins are also expressed in a variety of tumor cells, such as melanoma, prostate cancer, breast cancer, cervix carcinoma or pancreatic cancer.

While inhibition of integrin alphavbeta3 activity in endothelial cells has been studied extensively, any direct effects on integrin alphavbeta3 expressing tumor cells is hitherto unknown.

In this study we examined alphavbeta3 integrin expression in breast cancer cell lines, where by MDA MB 231 revealed high integrin alphavbeta3 as well as VEGFR-2 and VEGF expression.

While adhesion of MDA MB231 cells to an alphavbeta3 specific matrix such as fibrinogen was blocked by the addition of cyclic RGD peptides, sequentially addition of cyclic RGD peptides to already adherent MDA MB231 cells led to a diminished spreading in a time and dose dependent manner, starting at 20nM.

Furthermore, tumor cell migration as well as transmigration through an EC monolayer upon VEGF stimulation was blocked whenever alphavbeta3 integrins were targeted by RGD mimetic peptide.

Our data give first functional insights into the previously described effects of alphavbeta3 targeting small molecules, which led to a reduced breast cancer metastasis in vivo.

Thus interfering with alphavbeta3 integrins on tumor cells might represent a promising therapeutic lead in certain malignancies.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 424. doi:10.1158/1538-7445.AM2011-424
Abstract 4332: Conditional transgenic mice for the carboxyl-terminus of MUC16
Authors: Tian Huasong , Spriggs David R.
Published: June 2012

The cancer antigen 125 (CA125) is encoded by the MUC16 gene, which is present in sera of women with ovarian cancer. Currently, CA125 is widely used to diagnose the epithelial ovarian cancer and monitor the effects of treatment. Despite the widespread use of CA125, the biochemical and molecular functions of this antigen is poorly understood. There is no phenotype for MUC16 knockout mice (Cheon et al., PLoS ONE, 2009). Our previous data have demonstrated that the carboxyl-terminus of MUC16 has at least the partial functions of MUC16 since CA125 protein is too big (up to 2.5 MDa). To better understanding of the biological functions and characterizations of MUC16, we made a construct to create a conditional transgenic mouse model for the carboxyl-terminus of MUC16 using the Cre-loxP system. This mouse model encodes for the specific portion of MUC16, we named as MUC16ΔC354 mouse, which includes 354 amino acids of carboxyl-terminus of MUC16 with a HA tag. The CMV early enhancer plus chicken β actin (CAG) promoter is used to drive hGFP between two loxPs and the carboxyl-terminus of MUC16 after removing hGFP. After mating with EIIa-Cre transgenic mice, we found two founders which highly express MUC16ΔC354 in many organs, for example, brain, colon, heart, kidney, liver, lung, ovary and spleen. Lung and heart have higher expression level of MUC16ΔC354, and ovary has a middle level. MUC16ΔC354 heterozygous animals appear to be viable, fertile, and develop normally through the first four months of life. However, MUC16ΔC354 transgenic mice regulate PI3K/AKT and MAPK/ERK signaling pathways in some organs. Additional MUC16ΔC354 mice are being bred and selected for phenotypes. Cross breeding with N-Myc transgenic mice, or Rb-1 and p53 knockout mice is planned based on the preliminary results of The Cancer Genome Atlas (TCGA).

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4332. doi:10.1158/1538-7445.AM2011-4332
Abstract 4340: Sorafenib induces growth arrest and apoptosis in neuroblastoma cells via inhibition of JAK2/STAT3 and MEK1/2/MAPK (p44/42) signaling pathways
Authors: Yang Fan , Jove Veronica , Buettner Ralf , Xin Hong , Nam Sangkil , Ara Tasnim , DeClerck Yves A. , Seeger Robert C. , Yu Hua , Jove Richard
Published: June 2012

Neuroblastoma is the most common extracranial solid tumor in the pediatric population. It accounts for 6-10% of all childhood cancers. Currently, there is a critical need to find new drugs that are less toxic and target cell signaling pathways implicated as key mediators in the formation of neuroblastoma. Sorafenib (Nexavar), a multikinase inhibitor, blocks cell proliferation and induces apoptosis in certain types of cancers. Here, we tested antitumor effects of sorafenib (≤ 10 µM) on three human neuroblastoma cell lines, CHLA255, CHLA171 and SK-N-AS. Sorafenib inhibited cell proliferation and induced apoptosis in these neuroblastoma tumor cells in a time- and dose-dependent manner. Sorafenib inhibited phosphorylation of Signal Transducer and Activator of Transcription 3 (STAT3) proteins at Tyr705 in these cells as early as 15 min after treatment, associated with inhibition of phosphorylated JAK2, an upstream kinase that mediates STAT3 phosphorylation. Expression of a constitutively-activated STAT3 mutant (pSTAT3-C) partially blocked the antitumor effects of sorafenib on neuroblastoma cells. Sorafenib also inhibited the phosphorylation of STAT3 induced by IL-6 in these tumor cells. Moreover, sorafenib downregulated phosphorylation of MAPK (p44/42) in these neuroblastoma cells after 5 min treatment, consistent with inhibition of their upstream regulators MEK1/2. Finally, sorafenib inhibited expression of cyclin E, cyclin D1, cyclin D2 and cyclin D3, key regulators for cell cycle, and the antiapoptotic protein Mcl-1, which was accompanied by induction of growth arrest and apoptosis in neuroblastoma cells. Taken together, these findings suggest the potential use of sorafenib for the treatment of pediatric neuroblastomas.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4340. doi:10.1158/1538-7445.AM2011-4340
Abstract 4345: AZD8055, a combined TORC1/TORC2 inhibitor regulates Mycn protein expression and prevents neuroblastoma growth in vitro and in vivo
Authors: Vaughan Lynsey S. , Barker Karen , Webber Hannah , Jamin Yann , Robinson Simon , Guichard Sylvie , Chesler Louis
Published: June 2012

Neuroblastoma is the most common and deadly solid tumour of childhood (Brodeur, 2003). Amplification of the MYCN oncogene occurs in 25% of NBs correlating with poor clinical outcome. High risk neuroblastoma responds to current therapies but rapidly acquires chemoresistance; therefore alternative treatment strategies are required. Because of the importance of MYCN amplification in high risk neuroblastoma and specific expression of Mycn protein in tumour tissue, we have examined indirect methods of destabilising Mycn by targeting key components of upstream signalling pathways, responsible for maintenance of Mycn protein levels. Mycn protein stability is maintained by the RAS (RAF/MEK/ERK) and PI3K (PI3K/AKT/mTOR) pathways. The mammalian target of rapamycin (mTOR) plays an important role in cell proliferation, metabolism and tumourigenesis, and is frequently altered in cancer. Rapamycin and its analogues target the TORC1 complex and are effective against tumour xenografts but are limited by consequent up regulation of PI3K activity. Inhibition of TORC1 alleviates a TORC1 dependent negative feedback loop, with resulting activation of PI3K and AKT-dependent survival pathways.

AZD8055 is an mTOR kinase inhibitor that inhibits both TORC complexes with IC50 values in the nanomolar (nM) range. Treatment of N-myc wild-type or phosphomutant expressing neuroblastoma cell lines with AZD8055 inhibited mTOR-dependent phosphorylation of key pathway components, coincident with selective inhibition of wild-type but not phospho-mutated N-myc protein. Cell proliferation was reduced and sensitive cells accumulated in G1 as shown by flow cytometric analysis. Cells expressing N-myc phosphomutant proteins showed less sensitivity to AZD8055, with GI50 values above mechanistic range for targeted effects of this drug.

The ability of AZD8055 to inhibit neuroblastoma tumour progression was tested in a transgenic model of neuroblastoma (TH-MYCN) that over expresses Mycn protein and is predisposed to spontaneous tumour formation (Weiss, 97). We present data showing that AZD8055 inhibits tumour progression, reduces tumour cell proliferation and targets the mTOR complex in vivo.

In conclusion, inhibition of mTOR signalling is highly effective against MYCN-driven cell lines and tumours, and therefore could be a promising therapeutic strategy for targeted treatment of MYCN-amplified neuroblastoma.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4345. doi:10.1158/1538-7445.AM2011-4345
Abstract 4365: Glucosylceramide synthase promotes cancer stem cells accumulating in chemotherapy
Authors: Gupta Vineet , Bhinge Kaustubh N. , Liu Yong Yu
Published: June 2012

Cancer stem cells initiate tumorigenesis and are the causes of metastasis and relapse. Cancer stem cells, rather than other differentiated cells in tumors display resistance to cytotoxicity of anticancer drugs, but we do not know whether by-product of chemotherapy can induce cancer stem cells leading to chemotherapy failure. Glucosylceramide synthase (GCS) is a limiting enzyme regulating the synthesis of glycosphingolipids that play an essential role in the maintenance of embryonic stem cells. The purpose of this study was to identify and characterize the effect of ceramide glycosylation in the formation and maintenance of breast cancer stem cells (BCSCs). We have found that GCS overexpression was interrelated to the increment BCSCs and drug resistance in human breast cancer MCF-7 cell lines after doxorubicin induction. In MCF-7/Dox (doxorubicin-resistant) cells, GCS protein was increased by 39-fold accompanied with enhanced enzyme activity, as compared to wild-type MCF-7 cells. The BCSCs with CD24-/CD44+/ESA+ phenotype was increased by 5-fold in MCF-7/Dox cells as compared to MCF-7 cells. Silencing GCS by using mixed-backbone oligonucleotide (MBO-asGCS) significantly decreased the numbers of BCSCs more than 2-fold in MCF-7/Dox cells. In the soft agar colony formation assay, MCF-7/Dox cells showed significantly higher colonies formed as compare to the MCF-7 cells. BCSCs sorted from MCF-7/Dox cells displayed significantly higher GCS enzyme activity more than 3-fold and formed 2-fold more colonies, as compare with other non-stem cell subsets. The BCSCs cells showed significantly higher tumorigenicity and metastasibility as compared to non-stem cells (CD24-/CD44-) in athymic nude mice. More interestingly, doxorubicin treatment (1 mg/kg, once a week, and 40 days) considerably increased BCSCs 2-fold in tumors, and MBO-asGCS treatment eliminated the tumor growth and reduced metastasis due to reduction of BCSCs more than 2-fold in vivo. Comparison of glycosphingolipids and gene profile of stem cells indicated GCS or globo-series glycosphingolipids upregulates FGF1, FGFR1, ALDH2, ISL1, MSX1, SIGMAR1, PPARD and downregulates CXCL12, FRAT1, CCND1 to accumulate BCSCs during the course of chemotherapy. These results, for the first time, demonstrate that ceramide glycosylation by GCS accumulates BCSCs formation and causes aggressive tumor progression. Silencing of GCS that eliminates BCSCs can prevent and treat drug-resistant tumors and relapse.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4365. doi:10.1158/1538-7445.AM2011-4365
Abstract 4372: Radiation induces Notch-dependent de novo generation of breast cancer stem cells via expression of OCT-4/SOX2/NANOG
Authors: Lagadec Chann H. , Della Donna Lorenza , Vlashi Erina , Dekmezian Carmen , Brocks Tania , Pajonk Frank
Published: June 2012

Like in normal tissues, the self-renewal of cancer stem cells (CSCs) might be also under tight control of developmental pathway like the Notch, Wnt, Sonic Hedgehog or TGFβ pathways. The Notch pathway plays an important role in normal breast development, cell fate, and normal stem cell self-renewal, and its deregulation has been shown to play a role in cancer. Aberrant Notch signaling has been implicated in the development and progression of both preinvasive ductal carcinomas in situ and invasive breast cancers. Interestingly, in breast cancer, the Notch pathway plays major role for CSCs maintenance. We previously published that BCSCs (Breast Cancer Stem Cells) are relatively resistant to radiation and we demonstrated a link between BCSCs radio-resistance and the Notch pathway. Irradiation of MCF-7 and T47D cells increased the number of CSCs and this correlated with the induction of Notch signaling protein expression in a dose- and time-dependent manner. Jagged 1 expression was quickly (1h) increased 28-fold after 2Gy, DLL1 expression was increased 15-fold, 3 to 6h after irradiation with 2 or 4 Gy, Notch 2 expression was increased 16-fold 6h after 2, 4, 6 or 8 Gy, while DLL3 was increased 10-fold after the highest doses (6 to 12 Gy). Inhibition of Notch signaling pathway by a γ-secreatase inhibitor prevented enrichment for CSCs and reduced radiation-induced Notch protein expression. Most importantly, we demonstrated that radiation-induced Notch signaling contributes to the phenotype plasticity of BCSCs and their progeny. Using 2 different cancer stem cell markers (low proteasome activity and ALDH1) to found that radiation causes de novo generation of induced BCSCs (iBCSCs) from non-tumorigenic cells and this correlated with radiation-induced the expression of OCT-4, SOX2, and NANOG, key transcription factors of used to generate induced pluripotent stem cells (iPS cells).

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4372. doi:10.1158/1538-7445.AM2011-4372
Abstract 4382: Matricellular protein Cyr61/CCN1 regulates the stemness of oral cancer stem-like cells
Authors: Chang Chih-Hao , Leu Shih-Jeng , Lo Jeng-Fan
Published: June 2012

Background: Cysteine-rich 61 (Cyr61 [CCN1]), a matricellular and plasma membrane protein that has disparate function dependent on cell type-specific manner, is closely related to proliferation, invasion, angiogenesis and epithelial-mesenchymal transition (EMT). Besides, EMT promotes stemness properties in normal and neoplastic cells. Therefore, we want to examine whether CCN1 plays a role in our previous enriched oral cancer stem-like cells (OC-SLCs).

Methods: Initially, the expression of CCN1 in OC-SLCs translationally was evaluated. Further, the cell surface CCN1 positive cells from oral cancer cells or OC-SLCs were sorted for further study. We will compare the difference of stemness properties and tumorigenicity between CCN1 positive and negative cells. Further, downregulation of CCN1 in oral cancer cells was achieved to evaluate the function of CCN1 on oral cancer tumorigenesis in vitro and in vivo. The molecular mechanisms mediated by CCN1 on regulating the oral cancer tumorigenicity will also be elucidated

Results: We found that CCN1 protein was upregulated in OC-SLCs. Comparing to CCN1 negative cells, CCN1 positive cells exhibited higher sphere formation ability and highly expressed stem cell markers, Nanog and Oct-4. Flow cytometry analyses showed that cytoplasmic membrane CCN1 positive OC-SLCs also co-expression with cancer stem-like cell makers, ABCG2, Grp78 and CD133. In addition, CCN1 knockdown cells decreased sphere formation ability and displayed morphological alternation with loss of EMT. Analysis of CCN1 knockdown cells found that EMT related markers were significantly changed. Overall, we demonstrated that CCN1 is involved in maintenance of stemness and acquisition of epithelial-mesenchymal transition in OC-SLCs.

Conclusion: Our data suggest that Cyr61 could be a potential oral cancer stem cell marker, and also the key molecule to regulate the stemness of oral cancer stem-like cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4382. doi:10.1158/1538-7445.AM2011-4382
Abstract 4383: Regulation of cancer cell differentiation in vivo by delta-like 1 homolog (Drosophila) DLK1
Authors: Begum Asma , Kim Yuri , Lin Qun , Yun Zhong
Published: June 2012

Malignant tumor progression from a benign growth to metastasis is often accompanied by an accumulation of multiple genetic and epigenetic changes occurring over a course of more than a decade. The stem cell-like characteristics, especially the self-renewal capacity, of tumor cells is essential for uninterrupted inheritance of these disparate genetic and epigenetic changes in the same originating tumor cell. Like normal stem cells, cancer cells can lose their stemness or self-renewal potential in response to environmental stresses. Therefore, tumor cell stemness needs to be actively maintained. However, the underlying mechanisms remain to be clearly understood. Our previous studies and others have suggested that the tumor microenvironment, including hypoxia, plays a significant role in the regulation of cancer stem cell characteristics. We have identified a previously uncharacterized stem cell pathway mediated by DLK1 or delta-like 1 homolog (Drosophila) that is upregulated by hypoxia and enhances cancer cell stemness and tumorigenicity in neuronal tumors. In this study, we investigated the role of DLK1 in the regulation of cancer cell differentiation in vivo using neuroblastoma (NB) xenografts as a model. Our findings have revealed that overexpression of DLK1 promotes tumor growth by increasing the number of mitotic or proliferative cells and inducing angiogenesis. On the other hand, tumors derived from NB cells with down regulation of DLK1 expression by RNA interference or with overexpression of dominant negative mutants of DLK1 sensitize NB cells to undergo differentiation, as shown by increased expression of neuronal and glial differentiation markers. These results demonstrate that DLK1 plays an important role in the maintenance of undifferentiated, stem cell-like phenotype of NB cells in vivo. Our findings also indicate that the tumor microenvironment, especially hypoxia, can exert a profound impact on the tumor cell fate in vivo by regulating specific stem cell pathways.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4383. doi:10.1158/1538-7445.AM2011-4383
Abstract 4391: OCTN1- and OCTN2-mediated uptake of oxaliplatin in rat dorsal root ganglion neurons and HEK293 cells over-expressing OCTN1 or OCTN2
Authors: Jong Nancy N. , Nakanishi Takeo , Liu Johnson J. , Tamai Ikumi , McKeage Mark J.
Published: June 2012

Purpose: Organic cation transporters (OCTs) have been recently implicated in the uptake and toxicity of oxaliplatin. Peripheral neuropathy is the dose-limiting toxicity of oxaliplatin due to accumulation of platinum in the dorsal root ganglia (DRG) and damage to sensory neurons. This study sought to determine the role of OCTs in the DRG neuronal uptake of oxaliplatin.

Experimental Design: [14C] Oxaliplatin uptake and cytotoxicity in HEK/rOctn1, HEK/rOctn2, HEK/hOCTN1, HEK/hOCTN2, and primary culture of DRG neurons was determined to evaluate the role of Octn1 and Octn2 in oxaliplatin transport and neurotoxicity. mRNA, protein and functional expression of organic cation transporters in rat lumbar DRG were characterised using RT-PCR, Western blotting and uptake of model OCT substrates [3H] MPP, [14C] TEA, [3H] ergothioneine and [3H] L-carnitine.

Results: HEK293 cells over-expressing rOctn1, rOctn2, hOCTN1 and hOCTN2 showed increased uptake and cytotoxicity of oxaliplatin compared to Mock-transfected HEK293 controls, and Octn2-mediated uptake of oxaliplatin was significantly higher than Octn1 in both human and rat. Uptake of ergothioneine mediated by Octn1, and L-carnitine mediated by Octn2, was decreased during oxaliplatin exposure. Rat DRG showed high Octn1 and Octn2 mRNA levels, and uptake of ergothioneine and L-carnitine that was temperature- and sodium-dependent and significantly inhibited by model substrates of Octn1 and Octn2. In contrast, DRG had negligible functional activity and low or undetectable mRNA levels of Oct1, Oct2, and Oct3. [14C] Oxaliplatin uptake by DRG neurons was temperature- and sodium-dependent and inhibited by ergothioneine and L-carnitine. Ergothioneine and L-carnitine uptake by DRG neurons was inhibited in the presence of oxaliplatin.

Conclusions: In conclusion, Octn1 and Octn2, but not Oct1, Oct2 or Oct3, are functionally expressed in rat DRG neurons. Oxaliplatin uptake by rat DRG neurons and HEK293 cells was mediated by Octn2 and to lesser extent by Octn1.

Supported by Grant-in-Aid for Scientific Research (B) (I. T.), Research Activity Start-up (T. N.), Auckland Medical Research Foundation (Doctoral Scholarship) and Cancer Society of New Zealand (Research Grant).

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4391. doi:10.1158/1538-7445.AM2011-4391
Abstract 4404: Post-transcriptional regulation of dihydropyrimidine dehydrogenase (DPD) by the microRNAs mir27a and mir27b
Authors: Offer Steven M. , Wegner Natalie J. , Diasio Robert B.
Published: June 2012

The pyrimidine analog 5-fluorouracil (5-FU) and its prodrug capecitabine are prescribed widely for the management of several common cancers, including colorectal, breast, and pancreatic cancers. 5-FU is activated by conversion to cytotoxic metabolites via the pyrimidine anabolic pathway. In the opposing catabolic pathway, dihydropyrimidine dehydrogenase (DPD, encoded by the DPYD gene) initiates rapid and irreversible conversion of greater than 80% of administered 5-FU to inactive metabolites and limits the pool of 5-FU available for anabolism. Deficiency of DPD has been associated with adverse effects to 5-FU treatment, including mucositis, severe diarrhea, neurotoxicity, and life-threatening hematological toxicity, making DPD activity a potentially important predictor of 5-FU toxicity. Despite considerable study of the DPYD gene and its promoter, little is known about the post-transcriptional regulation of DPD expression at the molecular level. The objective of this study was to determine if DPD expression undergoes microRNA-mediated regulation. Computational analysis of the DPYD 3’ untranslated region (UTR) revealed an evolutionarily conserved seed-binding region for the microRNAs mir27a and mir27b. This finding prompted the hypothesis that both mir27a and mir27b are capable of modulating DPD expression. To determine if mir27a and/or mir27b bind to the DPYD 3’ UTR, HCT116 cells lines were engineered to stably express luciferase reporter constructs wherein the luciferase gene was modified to contain the 3’ UTR sequence from DPYD. As controls, stable cells lines containing mutations in the putative microRNA-binding region were generated concurrently. Two colon cancer cell lines (HT29 and HCT15) were also utilized to determine if mir27a and/or mir27b could modulate endogenous DPD expression. In cell lines expressing the luciferase gene juxtaposed to the DPYD 3’UTR, mimics of mir27a or mir27b resulted in significantly reduced luciferase activity relative to scramble control microRNA. Luciferase levels in mir27a or mir27b transfected lines were also significantly reduced compared to stable luciferase-expressing cells harboring the mutant UTR construct. Over-expression of small RNA inhibitors directed toward mir27a or mir27b led to increased luciferase activity in cells containing the wildtype UTR construct. Finally, over-expression of mir27a or mir27b significantly reduced endogenous DPD expression in both HT29 and HCT15 cells. Based on these findings, we conclude that mir27a and mir27b are capable of post-transcriptional regulation of DPD. To our knowledge this is the first report to show microRNA-mediated modulation of DPD expression.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4404. doi:10.1158/1538-7445.AM2011-4404
Abstract 4420: Analysis of functional role of interleukin-6 secreted by tumor-associated macrophages in ovarian cancer ascites
Authors: Isobe Aki , Sawada Kenjiro , Ooyagi Chifumi , Mabuchi Seiji , Wakabayashi Atsuko , Morishige Ken-Ichirou , Kimura Tadashi
Published: June 2012

Tumor-associated macrophages (TAMs) are known to contribute to tumor growth, invasion, and metastasis by producing various mediators. However, few studies have clarified the role of TAMs in human ovarian cancer patients. The aim of the present study was therefore to analyze the effect of TAM on ovarian cancer growth, invasion, and metastasis. Ascites were collected aseptically, and CD11b positive cells (TAMs) were purified by positive selection using magnetic-activate cell sorting (MACS) technology. Co-culture of ovarian cancer cell line, SKOV3ip1 cells, with TAMs significantly enhanced the invasive activity of cancer cells in Matrigel Boyden chamber assay. This enhancement was partially blolcked by the treatment of anti-interleukin (IL)-6 receptor antibody (Tocilizumab). Since SKOV3ip1 cells did not express IL-6 in mRNA and protein level, IL-6 secreted by TAMs were considered to play a key role in ovarian cancer invasion. In vivo ovarian cancer xenografts, i.p. treatment of Tocilizumab significantly reduced peritoneal dissemination and ascites formation. Those results suggested targeting IL-6 produced by TAMs could be a promising therapy for a subset of ovarian cancer patients.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4420. doi:10.1158/1538-7445.AM2011-4420
Abstract 4421: Curucmin suppresses myeloid-derived suppressor cells and inhibits gastric cancer growth
Authors: Tu Shuiping , Jin Huanyu , Shi Jingdong , Suo Ya , Liu Anna , LU Gang , Yang Chung S.
Published: June 2012

Myeloid-derived suppressor cells (MDSCs) are accumulated in the spleen and tumor and contribute to tumor growth, angiogenesis and progression. In this study, we examined the effects of curcumin, a compound from turmeric, on MDSCs and gastric cancer growth in vivo. BABL/C nude mice injected subcutaneously with gastric cancer MKN-45 cells were fed with control diet or 2% curcumin diet or received curucmin through daily i.p. injection at 50mg/kg body weight. Cell apoptosis were determined by Flow cytometer and TUNEL staining. Protein expressions were determined by Western Blot and Immunohistochemistry staining. The frequencies of myeloid-derived suppress cells in the mice were analyzed by Flow cytometer. Cytokines were determined by ELISA. Tumors growth was significantly inhibited by both 2% curucumin diet and curcumin i.p. injection treatment. The frequencies of MDSCs in the spleen, blood and tumor tissues were significantly decreased in the tumor-bearing mice after a 5-days or a 4-weeks treatment with curcumin (in diet or by i.p. injection) compared to control treatment. MDSCs isolated from the spleen of curcumin-treated mice secreted lower levels of IL-6, compared to that from untreated mice. The treatment of MDSCs isolated from the spleen of the untreated tumor-bearing mice with curcumin had lower levels of IL-6, compared to that of control mice. Furthermore, curcumin treatment significantly reduced IL-6 level secreted by IL-1beta-induced MDSCs. Curucmin treatment also significantly induced apoptosis of gastric cancer cells in vitro and in vivo. Our results demonstrate for the first time that cucumin could inhibit MDSCs mobilization and activation and that curcumin inhibits gastric cancer partly through suppressing MDSCs mobilization and activation. MDSCs may be a new target of cucumin for gastric cancer therapy and prevention. (The project was supported by NIH grant R21CA149865 (Tu SP)).

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4421. doi:10.1158/1538-7445.AM2011-4421
Abstract 4441: Intracellular delivery of therapeutic siRNA via an antennapedia-RNA-binding domain fusion peptide
Authors: Tsimon Myrsini , Kang Angray , Markiv Anatoliy , Kousparou Christina , Epenetos Agamemnon
Published: June 2012

A key characteristic in tumorigenesis is the aberrant expression of various oncogenes, whose cellular products drive various anti-apoptotic/pro-survival pathways. The RNAi pathway is an endogenous cellular pathway that transiently inhibits the translation of mRNA into protein. It therefore has the potential to selectively arrest the production of mutated proteins.

As the Intracellular delivery of bioactive molecules is still a major challenge, we are exploring the possible ability of the Antenapedia homeodomain (Antp) to act as an effective transporter of conjugated cargo across biological membranes. We have previously shown that Antp delivers both p21 into p21-null tumors in order to restore cell-cycle regulation, as well as a truncated form of MAML to inhibit Notch activation and induce apoptosis.

To test whether in silico-designed siRNA has the ability to silence mutated forms of genes, we have created a bifunctional fusion protein, TR7, which incorporates Antp and an RNA-binding protein. TR7 attempts to both neutralize the anionic nature of siRNA as well as introduce it to the cytoplasm, thus initiating the RNAi process in a non-cytotoxic manner.

In summary, this project, utilizing Antp, aims to solve the issue of systemic RNAi delivery, as well as harness the endogenous RNAi pathway to effectively halt the production of abnormal proteins in cancer and other life threatening illnesses.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4441. doi:10.1158/1538-7445.AM2011-4441
Abstract 4445: Novel antibody-dendrimer conjugates efficiently complex plasmid DNA
Authors: Wang Yang , McBride Bill , Goldenberg David M. , Chang Chien-Hsing
Published: June 2012

Introduction: Cationic polymers, such as polylysine, polyethylenimine, or polyamidoamine (PAMAM)-based dendrimers, form complexes with nucleic acids. However, their potential applications in nanomedicne as non-viral vectors for delivering therapeutic genes or siRNAs remain a challenge. One approach to improve selectivity and potency of a dendrimeric nanoparticle may be achieved by conjugation with an antibody that internalizes upon binding to target cells. In this study, we describe the synthesis and initial characterization of a novel immunoconjugate, designated E1-G5/2, which was made by the modular Dock-and-Lock (DNL) method to comprise half of a generation 5 (G5) PAMAM dendrimer (G5/2) site-specifically linked to a stabilized dimer of Fab derived from hRS7, a humanized antibody that is rapidly internalized upon binding to the Trop-2 antigen expressed on various solid cancers. Methods: E1-G5/2 was prepared by combining two self-assembling modules, AD2-G5/2 and hRS7-Fab-DDD2, under mild redox conditions, followed by purification on a Protein L column. To make AD2-G5/2, we derivatized the AD2 peptide with a maleimide group to react with the single thiol generated from reducing a G5 PAMAM with a cystamine core and used reversed-phase HPLC to isolate AD2-G5/2. We produced hRS7-Fab-DDD2 as a fusion protein in myeloma cells. The molecular size, purity and composition of E1-G5/2 were analyzed by size-exclusion HPLC, SDS-PAGE, and Western blotting. The biological functions of E1-G5/2 were assessed by binding to an anti-idiotype antibody against hRS7, a gel retardation assay, and a DNase protection assay. Results: E1-G5/2 was shown by size-exclusion HPLC to consist of a major peak (>90%) flanked by several minor peaks. The three constituents of E1-G5/2 (Fd-DDD2, the light chain, and AD2-G5/2) were detected by reducing SDS-PAGE and confirmed by Western blotting. Anti-idiotype binding analysis revealed E1-G5/2 contains a population of antibody-dendrimer conjugates of different size, all of which are capable of recognizing the anti-idiotype antibody, thus suggesting structural imperfections in the commercial supply of the G5 dendrimer. Gel retardation assay showed E1-G5/2 was able to maximally condense plasmid DNA at a charge ratio of 6:1 (+/-), with the resulting dendriplexes completely protecting the complexed DNA from degradation by DNase I. Conclusion: The DNL method can be used to build dendrimer-based nanoparticles that are targetable with antibodies. Such agents may have improved properties as carriers of drugs, plasmids, or siRNAs for diverse applications in vitro and in vivo.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4445. doi:10.1158/1538-7445.AM2011-4445
Abstract 4455: Influence of convective fluid flow on the penetration of anti-cancer drugs through multicell layers
Authors: Phillips Roger M. , Makeen Hafiz , Periasamy Raj , Loadman Paul M. , Jones Pamela F. , Sleeman Brian D. , Smye Stephen W. , Twelves Chris
Published: June 2012

One consequence of elevated interstitial fluid pressures (IFP) within solid tumours is disruption to the normal process of convective fluid flow (CFF) from the vasculature into the interstitium. Loss of convective fluid flow in tumours has a significant effect on reducing drug delivery and is recognised as a major factor that contributes to the drug resistant phenotype of many solid tumours. Experimental models of IFP and CFF have traditionally relied upon in vivo tumour models but here, we describe an in vitro assay which could be used to evaluate strategies designed to manipulate convective fluid flow. The model is based upon a conventional transwell model for assessing drug penetration across multicell layers which has been modified to allow the fluid level in top chamber to be raised above that in the bottom chamber. The difference in hydrostatic pressure across the multicell layer drives convective fluid flow, the rate of which can be varied by increasing or decreasing the height of fluid in the top chamber and/or by using multicell layers of different thicknesses. When CFF was 0.19 ml/min, the penetration of gefitinib, imatinib and doxorubicin across DLD-1 human colon carcinoma multicell layers was significantly enhanced compared to similar experiments where CFF was zero. The enhancement of penetration rates was 75 fold for doxorubicin, 53 fold for gefitinib and 62 fold for imatinib. The magnitude of these effects has potential therapeutic significance and support the proposal that strategies aimed at reducing tumour IFP and increasing CFF may enhance the efficacy of chemotherapy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4455. doi:10.1158/1538-7445.AM2011-4455
Abstract 4465: Inhibition of tumor growth, lymphangiogenesis, and metastasis by iron chelators, deferoxamine and deferasirox, in breast cancers
Authors: Kang Han Na , Kang Myoung Hee , Km Jung Lim , Kim Jun Suk , Choi Chul Won , Seo Jea Hong , Yoo Young A
Published: June 2012

Iron participates in a range of reactions that are necessary for cell viability and cell proliferation and it has been reported that iron depletion by chelator inhibits the proliferation of various cancer cells. Among the different kind of iron chelators, deferoxamine is the most widely studied molecule as an anti-tumor agent. Deferasirox is a new rationally-designed oral iron chelator and there are few reports regarding in vitro and in vivo effect on myeloid leukemia cells.This study was undertaken to evaluate the in vitro and in vivo effects of DFX (deferoxamine) and DFS (Deferasirox) on anti-cancer activities in breast cancer cells and to define molecular mechanisms underlying these effects. We found that DFX or DFS displayed significant anti-proliferative activity in a dose-and time-dependent manner in MDA-MB-231 and MDA-MB-435 cells. DFX or DFS treatment led to the enhanced percentages of apoptotic cell, which was mediated through the mitochondrial death pathway that requires up-regulation of Bad phosphorylation, PUMA, and caspase-3 /-9 activities. In addition, DFX or DFS treatment significantly attenuated the motility and invasiveness of metastatic breast cancer cells, which is attributed to the decrease in EMT process and lymphangiogenesis. Importantly, the in vivo study revealed that DFX or DFS retarded tumor growth and suppressed the metastatic capacity to lymph nodes by down-regulating lymphangiogenesis in the orthotropic breast tumor model. These data demonstrate that DFX or DFS induces anti-cancer activity of breast cancer cells by both inducing apoptosis and suppressing metastasis in vitro and in vivo. Hence, DFX and DFS could provide another strategy for treating breast cancers.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4465. doi:10.1158/1538-7445.AM2011-4465