bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 229: Identification and functional characterization of p130Cas as a substrate of protein tyrosine phosphatase non-receptor type 14
Authors: Zhang Peng , Wang Zhenghe
Published: August 2012

Protein tyrosine phosphatases (PTPs) are thought to be potentially important regulators in tumorigenesis as antagonists of protein tyrosine kinases (PTKs). Among them, Protein tyrosine non-receptor type 14 (PTPN14) has been shown to be mutated in both colorectal and breast cancers. However, little is known about the cellular signaling pathways it regulates. In order to determine whether PTPN14 has causative roles in tumorigenesis, we set out to identify substrates of PTPN14 and shed light on the mechanism why it is mutated in different tumor types. In this study, we report the identification of p130Cas, a scaffolding molecule, as a substrate of PTPN14 through a proteomic approach. We show that PTPN14 specifically dephosphorylates p130Cas at tyrosine 128 by shRNA knockdown and overexpression. We also demonstrate p130Cas is a direct substrate of PTPN14 by substrate trapping and in vitro phosphatase assays. We then mutate tyrosine 128 residue to phenylalanine in DLD1 and RKO, two different colorectal cell lines, to study the biological functions of this phosphosite. The mutant cells show reduced tumorigenicity based on colony formation and soft agar assays. However, the phenotypes diverge between the mutant cells from the two cell lines when we test the effect of the mutation on tumor growth in xenograft model. RKO Y128F mutant cells display significantly reduced tumor volumes while DLD1 Y128F mutant cells do not. In addition, RKO Y128F mutant cells are less invasive than parental cells in Matrigel but DLD1 Y128F mutant cells are surprisingly more invasive. We find that AKT is not fully activated in both mutant cells, which is likely the cause of the common phenotypes. However, phospho-ERK is up-regulated in DLD1 mutant cells possibly due to a compensatory mechanism but not in RKO mutant cells since ERK is already constitutively active in RKO cells, which explains the discrepancies in phenotypes mentioned above. Last, Src is shown to be the kinase of p130Cas Y128. When the phosphorylation of p130Cas Y128 is surveyed in six commonly used colorectal cancer cell lines, the phosphorylation levels are found to be correlated with the sensitivities to Src inhibitor. Thus, p130Cas Y128 phosphorylation can be utilized as a prognostic marker for Src inhibitors that are already in clinical trials.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 229. doi:1538-7445.AM2012-229
Abstract 2291: Oncogenic hsa-miR-1323 down-regulates MYCN/ALK and reveals PAG1 as a novel tumor suppressor for neuroblastoma
Authors: Ghosh Rajib , Chen Zaowen , Patterson Danielle , Sikorski Denae N. , Kim Eugene S. , Shohet Jason M. , Gunaratne Preethi H.
Published: August 2012

MicroRNAs (miRNAs) are 22-24 nucleotides non-coding RNAs that can simultaneously influence the levels of multiple target genes and therefore have the potential to strongly silence cancer gene networks. MYCN and ALK are well established oncogenes implicated in the pathogenesis and progression of neuroblastoma. Using multiple web based algorithms we have identified potential tumor suppressor miRNAs that can control the pathogenesis and/or progression of neuroblastoma by down-regulating both MYCN and ALK. We found hsa-miR-1323 as a candidate tumor suppressor for neuroblastoma. Lipofectamine based mimic transfection and lentiviral vector based over-expression of miR-1323 in cell lines led to a decrease in MYCN and ALK transcripts as well as signals from luciferase reporter constructs carrying the 3′-UTR of MYCN or ALK validating MYCN and ALK as direct targets of miR-1323. However, unexpectedly for this predicted tumor suppressor microRNA, gain of function studies for miR-1323 in neuroblastoma cell lines demonstrated increased proliferation, colony formation and tumor growth of orthotopic xenograft tumors. As SiRNA against MYCN strongly limits neuroblastoma proliferation, our data suggests that miR-1323 targets an alternative tumor suppressor pathway which by-passes the growth suppressive effects of MYCN inhibition and drives proliferation. Further bio-informatics analysis of putative miR-1323 targets pointed to PAG1 (or Cbp) as a possible tumor suppressor target of miR-1323. The transmembrane adaptor protein PAG1 inhibits proliferation driven by the Src family of kinases (SFKs) through multiple mechanisms and is a known tumor suppressor in leukemia and non-small cell lung cancers. Importantly, analysis of PAG1 in clinical cohorts of neuroblastoma reveals that low expression of PAG1 strongly correlates with poor overall survival compared to patients with high levels of PAG1 expression (p <0.001). We have confirmed that miR-1323 targets PAG1 transcript via qRT-PCR assays, and this is associated with increased p-ERK levels in neuroblastoma cell lines. We therefore propose that miR-1323 mediated repression of PAG1 activates the Src-dependent oncogenic pathways, overriding its effect on MYCN or ALK, and contributing to the aggressive clinical behavior of high-risk neuroblastoma. These studies further demonstrate the complexity of microRNA functions in normal and cancer cells and mandate careful evaluation of putative ‘tumor suppressive’ microRNAs as therapeutic tools.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2291. doi:1538-7445.AM2012-2291
Abstract 2292: Tumor suppressive miR-124 targets androgen receptor and inhibits proliferation of prostate cancer cells
Authors: Shi Xu-Bao , Xue Lingru , Tepper Clifford G. , White Ralph W. deVere
Published: August 2012

Recent studies have revealed that miR-124 functions as a tumor suppressor and is downregulated in several types of human cancer. Until now, however, whether miR-124 is involved in prostate cancer (CaP) is totally unknown. In order to explore the role of miR-124 in CaP, we detected the expression level of miR-124 in prostate cell lines and clinical prostatic tissues using qPCR, and observed a reduced expression of miR-124 in all malignant cell lines tested and in a majority of clinical CaP samples examined, compared to that in the benign cell lines and in benign prostatic hyperplasia (BPH), suggesting that miR-124 is a putative tumor suppressor gene. Since the 3′UTR of androgen receptor (AR) mRNA contains a broadly conserved miR-124-binding site, experiments were performed to validate miR-124-mediated regulation of AR. We found that transfection of synthetic miR-124 mimic (miR-124m) downregulated the AR protein level and its downsteam effectors PSA and miR-125b. To verify that the miR-124 binding site is responsible for regulation by miR-124, the AR 3β UTR was cloned into the pRIM Reporter-luciferase vector, and cotransfected with miR-124m into CaP cells. We observed that transfection of miR-124m resulted in 40% reduction of luciferase activity, indicating a direct interaction between miR-124 and AR mRNA. We then examined whether clinical CaP samples having low level of miR-124 overexpress the AR using IHC analysis, and found a reverse correlation between miR-124 and AR expression levels in 7 of 8 CaP samples. These data suggest that miR-124 targets the AR and down-regulation of miR-124 results in increased expression of the AR in CaP cells. In addition, we tested whether miR-124 inhibits the growth of CaP cells, and found that treatment with miR-124m induced significant inhibition of cell proliferation compared to treatment with miR-NC control. We also evaluated the effect of miR-124 on tumorigenesis of 22Rv1 CaP cells that were transiently transfected with miR-124m. We observed that tumors derived from miR-124-transfected 22Rv1 cells grew substantially slowly, when compared to miR-NC tumors. Taken together, our data suggest that miR-124 is a potential tumor suppressor gene in CaP, and restoration of miR-124 expression may represent a novel therapeutic strategy in CaP therapy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2292. doi:1538-7445.AM2012-2292
Abstract 2293: MicroRNA-1826 targets VEGFC, beta-catenin (CTNNB1) and MEK1 (MAP2K1) in human bladder cancer.
Authors: Hirata Hiroshi , Hinoda Yuji , Ueno Koji , Shahryari Varahram , Tabatabai Z. Laura , Dahiya Rajvir
Published: August 2012

Introduction and Objective: The Wnt/beta-catenin (CTNNB1) and Ras-Raf-MEK-ERK signaling pathway play an important role in bladder cancer progression. Tumor suppressive miRNAs targeting these cancer pathways may provide a new therapeutic approach for bladder cancer. Methods: We initially identified miRNA-1826 potentially targeting CTNNB1, VEGFC, MEK1 using several target scan algorithms. The expression of miR-1826 was confirmed in bladder cancer tissues and bladder cancer cell lines. Then we performed several functional analyses of miR-1826. Finally 3′UTR luciferase assay and Western blot were performed to look at relationship between miR-1826 and target genes. Results: MiR-1826 expression was significantly lower in bladder cancer tissues and bladder cancer cell lines (J82, T24, TCC-SUP) compared to normal bladder tissues and normal bladder cell lines (SV-HUC-1). The expression of miR-1826 was lower in bladder cancer tissues and inverse correlation of miR-1826 with several clinical parameters (pT, grade) was observed. The functional analyses showed that miR-1826 inhibited bladder cancer cell viability, invasion and migration. Also we also found increased apoptosis and G1 cell cycle arrest in miR-1826 transfected bladder cancer cells. 3′UTR luciferase activity and protein expression of these target genes (CTNNB1, MEK1, VEGFC) were significantly down-regulated in miR-1826 transfected bladder cancer cells (J82, T24). To examine whether the effect of miR-1826 was through CTNNB1 (beta-catenin) or MEK1 knock down, we knocked down CTNNB1/MEK1 mRNA using a si-RNA technique. We observed that CTNNB1 or MEK1 siRNA knockdown resulted in effects similar to those with miR-1826 in bladder cancer cells. Conclusions: Our data suggests that the miR-1826 plays an important role as tumor suppressor via CTNNB1/MEK1/VEGFC down-regulation in bladder cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2293. doi:1538-7445.AM2012-2293
Abstract 2296: Aldose reductase inhibition prevents human colon cancer cells growth by miR-21 and FOXO3a-mediated regulation of PTEN
Authors: Srivastava Satish K. , Saxena Ashish , Ramana Kota V.
Published: August 2012

We have shown earlier that the inhibition of aldose reductase (AR), a NADPH-dependent aldo-keto reductase, prevents growth factor-induced proliferation of colon cancer cells in vitro and in vivo. Recent studies indicate that changes in microRNA (miRNA) expression can contribute to cancer by modulating the functional expression of critical genes involved in cancer growth and metastasis. However, the molecular mechanism(s) by which AR regulates miRNA expression and their dependent mitogenic effects in cancer cells is not known. Therefore, we investigated how AR regulates growth factor -induced expression of miRNAs in colon cancer cells. Our results indicate that inhibition of AR significantly down-regulated growth factor-induced miR-21 expression in HT29, SW480 and Caco-2 human colon cancer cells. Further, AR inhibition also increased PTEN, a direct target of miR-21 that contributes cancer cell growth and invasion. Inhibition of AR also prevented the EGF-induced phosphorylation of PTEN and FOXO3a, which prevents cell proliferation. Treatment of human colon cancer cells with AR inhibitor, fidarestat, prevented the EGF-induced DNA binding activity of AP1, phosphorylation of PI3K, AKT, c-Jun and c-Fos. More importantly, in HT29 human colon adenocarcinoma xenograft tissues, miR-21 expression was lower, and PTEN and FOXO3a levels were significantly higher in AR inhibitor-treated mice compared to controls. Collectively, these results show a novel role of AR in mediation of growth factor-induced colon cancer cell proliferation by increasing miR-21 expression and inactivating or decreasing PTEN and FOXO3a through the ROS/PI3K/AKT/AP1 pathway, indicating that AR inhibitors could be used for the prevention/therapy of colon carcinogenesis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2296. doi:1538-7445.AM2012-2296
Abstract 2297: MicroRNAs inhibit breast canncer metastasis via targeting CXC chemokine receptor 4
Authors: Liang Zhongxing , Huang Ke , Hong Samuel , Ahn Jeffrey , Shim Hyunsuk
Published: August 2012

Overexpressed CXC chemokine receptor (CXCR4) strongly contributes to breast cancer metastasis. However, the underlying mechanisms of CXCR4 overexpression need still to be explored. MicroRNAs (miRNA) have been implicated as a class of critical regulators controlling diverse biological processes. In the present study, we investigated whether miR-302 and miR-588 are involved in the regulation of CXCR4 and play a functional role in breast cancer metastasis. We have shown that miR-302 and miR-588 were downregulated in highly metastatic breast cancer cell lines and tissue specimens compared to low metastatic cell lines and breast cancer tissues. The decreased expression levels were inversely consistent with overexpression of CXCR4 in highly breast cancer cell lines and tissues. In addition, we found that restoration of miR-302 and miR-588 resulted in a dramatic reduction of CXCR4 expression at protein levels by directly targeting its 3′ untranslated region. Furthermore, the elevated expression of miR-302 and miR-588 significantly inhibited the invasion of breast cancer cells. More promisingly, our data have demonstrated that overexpression of miR-302 reduced metastasis and growth of breast cancer in vivo. Our findings describe a new mechanism for the regulation of CXCR4 and metastasis of breast cancer. The newly characterized miR-302/588/CXCR4 link provides potential molecular markers for predicting breast cancer metastasis. MiR-302 and miR-588 may have a therapeutic potential to suppress breast cancer metastasis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2297. doi:1538-7445.AM2012-2297
Abstract 2298: MiR-23a regulates TGF-β-induced epithelial-mesenchymal transition by targeting E-cadherin in pulmonary epithelium cells
Authors: Seike Masahiro , Mengru Cao , Soeno Chie , Noro Rintaro , Minegishi Yuji , Yoshimura Akinobu , Gemma Akihiko
Published: August 2012

Transforming growth factor-β (TGF-β)-induced epithelial-mesenchymal transition (EMT) has been reported to be related to pathogenesis of various diseases including lung cancer and idiopathic pulmonary fibrosis (IPF). Recently, microRNAs (miRNA) have been recognized as a new class of genes involved in human tumorigenesis. MiR-23a/24/27a is a miRNA cluster located in chromosome 19p13.12, which can function as an oncogene in several human cancers. In this study, we analyzed miR-23a/24/27a expression in 10 non-small cell cancer (NSCLC) cell lines by real-time PCR analysis. Correlation between expression of these miRNAs and TGF-β/Smad signaling was evaluated. We found that miR-23a could be regulated by TGF-β1 in a Smad-dependent manner in A549 lung adenocarcinoma cells originated by type II pneumocyte showing the EMT phenomenon. Knockdown of miR-23a partially restored E-cadherin expression under the condition of TGF-β1 stimulation. In contrast, overexpression of miR-23a could suppress E-cadherin expression and stimulate EMT. Furthermore, A549 cells with overexpressed miR-23a were more resistant to gefitinib than the parental cells. These findings suggested that miR-23a regulates TGF-β-induced EMT by targeting E-cadherin in pulmonary epithelium cells and may be useful as a new therapeutic target in pulmonary diseases including NSCLC and IPF.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2298. doi:1538-7445.AM2012-2298
Abstract 2299: Genistein suppresses prostate cancer growth through inhibition of oncogenic miR-151
Authors: Chiyomaru Takeshi , Zaman Mohd Saif , Majid Shahana , Deng Guoren , Yamamura Soichiro , Shahryari Varahram , Saini Sharanjot , Hirata Hiroshi , Ueno Koji , Chang Inik , Tanaka Yuichiro , Enikida Hideki , Nakagawa Masayuki , Dahiya Rajvir
Published: August 2012

Introduction and Objective: Genistein has been shown to suppress the growth of several cancer cell lines in vitro and in vivo, reducing the expression of several oncogenic microRNAs. However, the effects of genistein on the regulation of oncogenic miR-151 have not been reported. In this study, we investigated whether genistein could alter the expression of oncogenic miR-151 and its target genes that are involved in the progression and metastasis of prostate cancer. Methods: The expression levels of miR-151 were analyzed by TaqMan quantitative real-time PCR and normalized to RNU48. Nineteen clinical specimens were obtained from patients at the San Francisco Veterans Affairs Medical Center from 2000 to 2006. For gain-and loss-of-function assay, cell proliferation (MTS assay), migration (wound healing assay), and invasion (matrigel invasion assay) were evaluated in human prostate cancer cell lines, PC3 and DU145. Predicted target genes and their target microRNA binding site seed regions were investigated using TargetScan and microRNA.org. Genistein was administered every day along with a change of medium, and the cells were grown for four days. Results: Real-time RT-PCR showed that the expression of miR-151 was higher in PC3 and DU145 cells compared with RWPE-1 cells. Treatment of PC3 and DU145 cells with 25 μM genistein down-regulated the expression of miR-151 compared with vehicle control. Kaplan-Meier curves and log-rank tests revealed that high expression levels of miR-151 had a significant adverse effect on survival rate. Inhibition of miR-151 in prostate cancer cells by genistein significantly inhibited cell migration and invasion. By in-cilico analysis, several genes (N4BP1, CASZ1, IL1RAPL1, LGI1, SOX17 and TOM1L1) that are suggested to have tumor suppressive function in several types of cancers were detected as candidate target genes of miR-151. Conclusions: We demonstrated that genistein down-regulated miR-151 that functions as an oncogenic miRNA in prostate cancer. This study suggests that genistein mediated suppression of oncogenic miRNAs can be an important dietary therapeutic strategy for the treatment of prostate cancer. Source of Funding: Grants R01CA160079-01, R01CA138642, VA Research Enhancement Award Program (REAP), and VA Merit Review grants

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2299. doi:1538-7445.AM2012-2299
Abstract 230: A novel quantitative phosphoproteomic approach in mouse xenograft models to identify Src oncogenic signaling in colorectal cancer
Authors: Sirvent Audrey , Urbach Serge , Roche Serge
Published: August 2012

The non receptor tyrosine kinase (TyrK) Src mediates signal transduction induced by growth factors and integrins. Src also exhibits oncogenic activity when deregulated, a situation frequently observed in colorectal cancer (CRC). Moreover, the extent of kinase deregulation often correlates with malignant potential, in agreement with an important role Src in this tumorigenic process. However, the exact role of Src in CRC is only partially elucidated. Src has been implicated in growth and invasiveness of various CRC cells. A previous SILAC quantitative phosphoproteomic analysis allowed the characterization of Src signaling leading to CRC cells growth and invasion (Leroy et al, Cancer Res, 2009, 15:2279-86). Interestingly, Src also increased tumor growth in nude mice engraft with human SW620 CRC cells. This biological effect correlated with a strong increase of tyrosine phosphorylation (pTyr) content in Src-positive tumors. Here, we wished to characterize Src oncogenic signaling in these xenograft tumor models. To this end, we have developed an innovative quantitative phosphoproteomic analysis coupled to stable isotope labeling in mice named SILAX for Stable Isotope Labeling in mouse Xenografts. This method is based on tumor heavy isotope labeling in nude mice by addition of [13C]-Lysine into the food. An incorporation level >87% was obtained in subcutaneous tumors after 30 days of heavy lysine diet. This approach allowed the identification of 61 proteins which exhibited a significant increase in and/or association with pTyr upon Src expression in tumors. These mainly include vesicular trafficking, signaling and RNA binding proteins. Interestingly, we identified the novel vesicular trafficking protein TOM1L1 (Franco et al, Mol Cell Biol, 2006, 26:1932-47) as a specific regulator of Src oncogenic signaling of CRC cells in vivo. While TOM1L1 depletion poorly affected Src-induced CRC proliferation in vitro, it significantly inhibited tumor growth in nude mice. This data suggests that vesicular trafficking plays an important role in Src-induced CRC tumor growth and validate SILAX as a valuable approach to decipher TyrK signaling in mouse xenograft models.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 230. doi:1538-7445.AM2012-230
Abstract 2302: Epigenetic activation of miR-200a targets the Nrf2-dependent chemoprevention pathway in breast cancer
Authors: Zhou Qun , Yao Yuan , Gabriel Eades
Published: August 2012

NF-E2-related factor 2 (Nrf2) is an important transcription factor that activates the expression of cellular detoxifying enzymes. Nrf2 expression is largely regulated through Nrf2 association with Kelch-like ECH-associated protein 1 (Keap1), which results in cytoplasmic Nrf2 degradation. Conversely, little is known concerning the regulation of Keap1 expression. Until now, a regulatory role for microRNAs (miRs) in controlling Keap1 gene expression had not been characterized. By using miR array-based screening, we observed miR-200a silencing in breast cancer cells and demonstrated that upon re-expression, miR-200a targets the Keap1 3′ untranslated region (3′UTR) leading to Keap1 mRNA degradation. Loss of this regulatory mechanism may contribute to the dysregulation of Nrf2 activity in breast cancer. Previously, we have identified epigenetic repression of miR-200a in breast cancer cells. Here, we find that treatment with epigenetic therapy, the histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA), restored miR-200a expression and reduced Keap1 levels. This reduction in Keap1 levels corresponded with Nrf2 nuclear translocation and activation of Nrf2-dependent NAD(P)H-quinone oxidoreductase 1 (NQO1) gene transcription. Moreover, we found that Nrf2 activation inhibited the anchorage-independent growth of breast cancer cells. Finally, our in vitro observations were confirmed in a model of carcinogen-induced mammary hyperplasia in vivo. In conclusion, our study demonstrates that miR-200a regulates the Keap1/Nrf2 pathway in mammary epithelium and we find that epigenetic therapy can restore miR-200a regulation of Keap1 expression, therefore re-activating the Nrf2-dependent antioxidant pathway in breast cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2302. doi:1538-7445.AM2012-2302
Abstract 2307: MiR-365 regulates lung cancer and developmental gene TTF-1
Authors: Qi Ji , Rice Shawn J. , Salzberg Anna C. , Runkle Aaron , Liao Jason , Zander Dani S. , Mu David
Published: August 2012

Thyroid transcription factor 1 (TTF-1 or NKX2-1) is an essential fetal lung developmental factor which can be recurrently activated by gene amplification in adult lung cancer. We have discovered the first microRNA (i.e., miR-365) that directly regulates TTF-1 by interacting with its 3′-untranslated region. The finding that miR-365 is expressed in tissues beyond TTF-1 expression suggests the existence of other targets, which was substantiated by a subsequent gene expression profiling assay. In line with the microRNA/target relationship, the expression patterns of miR-365 and TTF-1 were in an inverse relationship in human lung cancer. Exploration of human lung cancer genomics data uncovered that TTF-1 gene amplification was significantly associated with DNA copy number loss at one of the two genomic loci encoding the precursor RNA of mature miR-365 (i.e., mir-365-1). This implies the existence of genetic selection pressure to lose the repressive miR-365 that would otherwise suppress amplified TTF-1. In studying how this microRNA/target relationship affects TTF-1 function in transforming growth factor beta-induced epithelial to mesenchymal transition (EMT), we detected a signaling loop between TGFbeta and miR-365 and this loop reinforced suppression of TTF-1 via miR-365. Surprisingly, mir-365 also targeted an EMT-promoting gene HMGA2, which may at least in part explain the difficulty for detection of EMT related phenotypes associated with miR-365. In summary, these data connect the lung transcriptional program to the microRNA network.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2307. doi:1538-7445.AM2012-2307
Abstract 2308: MUC1 modulation of E-cadherin through miR-200c and ZEB1
Authors: Mohr Ashley M. , Lewallen Michelle E. , Radhakrishnan Prakash , Liu Xiang , Bailey Jennifer M. , Hollingsworth Michael A.
Published: August 2012

MUC1, a cell-surface glycoprotein implicated in tumorigenesis and metastasis, has a cytoplasmic tail (MUC1.CT) that is phosphorylated by different receptor tyrosine kinases and conducts these signals by binding and altering the function or stability of transcription factors, thereby influencing gene transcription. MicroRNAs (miRNAs), short RNAs (19-22 nucleotides), regulate gene expression at the transcriptional level and are differentially regulated in cancer and metastasis. We discovered that MUC1.CT influences expression of miRNAs that contribute to metastasis, including miR-200c. MiR-200c is a known regulator of epithelial-to-mesenchymal transition (EMT) through regulation of ZEB1, a transcriptional repressor of E-cadherin. In addition, ZEB1 is a repressor of miR-200c in a feedback loop. Overexpression of MUC1 results in a dramatic decrease in miR-200c levels, resulting in increased levels of ZEB1. Chromatin immunoprecipitation (ChIP) was used to demonstrate that MUC1.CT occupies the ZEB1 binding motif upstream of the miR-200c start site. In addition, we found that ZEB1 occupancy of the miR-200c promoter region was enhanced 3.5-fold in MUC1 overexpressing cells, indicating that MUC1.CT may influence the expression and stability of ZEB1 at the miR-200c promoter. Co-immunoprecipitation experiments determined that MUC1.CT directly interacts with ZEB1. We evaluated levels of proteins affected by the miR-200c pathway in MUC1 overexpressing cells and observed an increase in ZEB1 levels and a decrease in E-cadherin levels, consistent with induction of EMT. We also confirmed an increase of ZEB1 occupancy at the E-cadherin promoter in MUC1 overexpressing cells, further confirming our hypothesis. In addition, MUC1.CT is differentially phosphorylated by several receptor tyrosine kinases. Antibodies against different phospho-isoforms were used with ChIP to investigate the signaling cascades that influence localization of the MUC1.CT to the miR-200c promoter. The phospho-YEKV form of MUC1.CT, which is representative of signaling through epidermal growth factor/receptor (EGF/EGFR), was the only phospho-MUC1.CT detected at the ZEB1 binding motif of the miR-200c promoter. EGF signaling is linked to a loss of E-cadherin, and our work is the first to link EGF signaling through the MUC1.CT as a direct regulator of miR-200c expression. These data portray a novel pathway of EMT modulation. EGF signaling via MUC1 regulates levels of miR-200c, altering regulation E-cadherin. Grant support was provided by grants from the NCI (Training Grant CA09476, R01CA57362, U01 CA111294) and student assistantships from UNMC.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2308. doi:1538-7445.AM2012-2308
Abstract 2314: Dovitinib demonstrates antitumor and anti-metastatic activities in xenograft models of hepatocellular carcinoma
Authors: Hung Huynh T. , Choo Su Pin , Soo Khee Chee , Ong Richard , Linnartz Ronald , Shi Michael Ming
Published: August 2012

Background and aims: Hepatocellular carcinoma (HCC) is the third leading cause of cancer death. Although sorafenib has been shown to improve survival of patients with advanced HCC, this improvement is modest and patients eventually have refractory disease. This study aims to investigate the antitumor, antiangiogenesis and antimetastatic activities of dovitinib in preclinical models of HCC. Methods: 21-0208 and SK-HEP1 cells as well as patient-derived HCC models were employed to study of the antitumor effect of dovitinib. Changes of biomarkers relevant to FGFR/VEGFR/PDGFR pathways were determined by Western blotting. Microvessel density, apoptosis and cell proliferation were analyzed by immunohistochemistry. Results: Treatment of SK-HEP1 cells with dovitinib resulted in G2/M cell cycle arrest, inhibition of colony formation in soft agar and blockade of bFGF-induced cell migration. Dovitinib inhibited basal expression and FGF-induced phosphorylation of FGFR-1, FRS2-α and ERK1/2. In vivo, dovitinib potently inhibited tumor growth of six HCC lines. Inhibition of angiogenesis correlated with inactivation of FGFR/PDGFR-α/VEGFR-2 signaling pathways. Dovitinib also caused dephosphorylation of retinoblastoma, upregulation of p-histone H2A-X and p27, and downregulation of p-cdk-2 and cyclin B1, which resulted in a reduction in cellular proliferation and the induction of tumor cell apoptosis. In an orthotopic model, dovitinib potently inhibited primary tumor growth and lung metastasis and significantly prolonged mouse survival. Conclusion: Dovitinib demonstrated significant antitumor and antimetastatic activities in HCC xenograft models. This study provides a compelling rationale for clinical investigation in patients with advanced HCC.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2314. doi:1538-7445.AM2012-2314
Abstract 2315: Pharmacodynamic and antitumor activity of fragment-derived inhibitors of MetAP2 in tumor xenografts
Authors: Smyth Tomoko , Curry Jayne , Wilsher Nicola , Johnson Chris , Berdini Valerio , Richardson Caroline , Massey Frances , McMenamin Rachel , Griffiths-Jones Charlotte , Thompson Neil , Wallis Nicola G.
Published: August 2012

Background: Methionine aminopeptidase (MetAP) 2 is one of the two eukaryotic enzymes responsible for cleaving the N-terminal methionine from newly synthesized polypeptides to allow further post-translational modifications such as myristoylation to take place. The enzyme is the target of the anti-angiogenic natural product, fumagillin and so is believed to play a role in angiogenesis. Fumagillin analogues have shown activity in several disease models including oncology, inflammation and obesity indicating MetAP2 is a promising target in a number of indications. The semi-synthetic fumagillin analogue, TNP470, has shown activity in a Phase I/II cancer trial, suggesting that MetAP2 is a good oncology target. Here we describe the discovery, optimization and anti-tumor activity of fragment-derived MetAP2 inhibitors. Results: We identified fragment hits to MetAP2 using our fragment-based screening approach, Pyramid™. These were optimized by structure-based drug design to novel, potent lead compounds with sub-100 nM potency against the isolated MetAP2 enzyme. The two most advanced compounds inhibited proliferation of HUVECs with potencies of 130 nM and 300 nM. Levels of the MetAP2 substrate, Met-14-3-3, were shown to increase on treatment of HUVECs with these compounds indicating that Met-14-3-3 was not being processed and hence MetAP2 was being inhibited. The compounds also inhibited HUVEC tubule formation demonstrating their anti-angiogenic properties. The two lead compounds were further tested in vivo. Both compounds were well tolerated at doses up to 200 mg/kg bid. Levels of Met-14-3-3 were seen to increase in the thymus and spleen (where high expression of MetAP2 has been reported) of mice treated with the compounds, indicating again that MetAP2 was being inhibited in these tissues. Compound 1 was tested in a mouse HCT116 xenograft model. Mice were subcutaneously inoculated with HCT116 cells and oral dosing at 200 mg/kg bid started one day later. Tumor growth was inhibited in treated mice compared with control and growth inhibition was greater in Compound 1 treated mice (T/C 46%) compared with mice treated with 30 mg/kg TNP470 subcutaneously q2d (T/C 61%). Conclusions: The compounds described here are novel small-molecule inhibitors of MetAP2. Their promising tumor growth inhibitory properties merit their testing in further tumor models and potentially other indications.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2315. doi:1538-7445.AM2012-2315
Abstract 2320: A multi-domain fusion molecule inhibits tumor growth affecting in-vivo angiogenesis
Authors: Boosani Chandra Shekhar , Gunda Venugopal , Verma Rajkumar , Yakkanti Sudhakar Akul
Published: August 2012

Growth of solid tumors beyond their benign size largely depends on the availability of oxygen and food supply which is accomplished through induced tumor angiogenesis. Inhibition of such de novo angiogenesis should aid in arresting the growth of solid tumors. Besides availability of several monoclonal antibodies and synthetic inhibitors for target molecules that induce tumor angiogenesis, endogenous antiangiogenic molecules are effective candidates with no drug resistance. Several endogenous angioinhibitors have been identified and were proven to inhibit tumor angiogenesis both in-vitro and in-vivo. However, many of them exhibited varied effects in inhibiting tumor growth with multiple mechanism(s) of action. Thus in our present study we developed a fusion antiangiogenic molecule “Combostatin” using the functional domains from two of the known antiangiogenic molecules reported by us. Here we show that Combostatin is an effective inhibitor of angiogenesis and inhibits tumor growth in-vitro and in-vivo. We also show that Combostatin affects several of the antiangiogenic pathways such as endothelial cell proliferation, migration and tube formation which are mediated by its binding to several integrin receptors on the endothelial cell surface. Our studies conclude that Combostatin is a more potent inhibitor of tumor angiogenesis than its parent molecules generated form type IV and Type XVIII collagen.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2320. doi:1538-7445.AM2012-2320
Abstract 2324: LHT-7 as multi-targeting and anti-angiogenic agent to growth factor receptors
Authors: Bae Sang Mun , Park Rang-Woon , Jung Yoonjae , Yoo-Jin Yang , Chi Lianhua , Byun Youngro
Published: August 2012

Angiogenesis is a highly regulated process that is critical for tumor growth and metastasis. Angiogenesis depends on the balance between different molecules released by the host and tumor cells such as vascular endothelial growth factor (VEGF), fibroblast growth factor basic (bFGF), platelet-derived endothelial cell growth factor (PDGF), insulin-like growth factors (IGF-1 and IGF-2) and scatter factor, which are potential targets for development of angiogenesis inhibitor and depend on heparin sulfate for their biological functions. Heparan sulfate is located in cell surface and in the extracellular matrix and plays crucial regulatory roles in some growth factors. For this reason, heparin has been highlighted as a therapeutic agent as well as anticoagulant. In a previous study, we reported new angiogenesis inhibitor, low molecular weight heparin derivative (heparin-taurocholic acid conjugate; LHT-7) and it showed delay of in vivo tumor growth in MDA-MB231 human breast cancer and inhibited phosphorylation of VEGFR2 through the interaction with VEGF in human umbilical vein endothelial cells (HUVECs). In present study, we revealed LHT-7 as multi-targeting agent to several growth factors and on their actions. LHT7 inhibited phosphorylation of VEGFR2 and binding of LHT7 was increased in VEGFR2 over-expressing HEK293 by FACS analysis. In HUVECs, binding of LHT-7 was shown in a dose-dependent manner by FACS analysis and co-localized with VEGFR2 by immunofluorescence. LHT-7 also inhibited vessel formation enhanced by PDGF-bb in matrigel plug assay and phosphorylation of PDGFR-ß in human vascular smooth muscle cell (HuVSMC). Binding of LHT-7 was increased in a dose-dependent manner by FACS analysis and decreased in PDGFR-ß-siRNA HuVSMC by immunofluorescence. Finally, LHT-7 inhibited phosphorylation of FGFR-1 in HUVECs and binding of LHT-7 was decreased in FGFR-1-siRNA MDA-MB-361. Overall, LHT-7 has inhibitory effect on phosphorylation of growth factors and binding of LHT-7 was increased in a dose-dependent manner but decreased in a growth factor down-regulated cell. Therefore we suggest that LHT7 would target to VEGFR2, PDGFR- ß and FGFR-1 and inhibit their angiogenic roles.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2324. doi:1538-7445.AM2012-2324
Abstract 2327: Anti-angiogenic activity of a CovX bi-functional antibody conjugate targeting both activin-receptor like kinase type 1 (ALK-1) and vascular endothelial growth factor (VEGF)
Authors: Lee Alice , Stock Joseph , Do Janet , Matin Marla , Rosario Joselyn Del , feng Lijin , Yamamoto Sachiko , Souratha Jennifer , Tchistiakov Lioudmila , Gallo Michael , Gao Yijie , Pirie-Shepherd Steven , Bissonnette Reid , Lappe Rodney , Woodnutt Gary , Blot Vincent
Published: August 2012

Angiogenesis is essential for tumor growth and metastasis, and controlling tumor-associated angiogenesis is a proven therapeutic intervention to limit tumor progression. Several pathways have been involved in tumor-associated angiogenesis and the predominant target of current interventions is the vascular endothelial growth factor (VEGF) pathway. Recently, Activin-receptor like kinase type 1 (ALK-1) has been proven to be a novel and potent target of tumor angiogenesis. Our results indicate that inhibiting VEGF and ALK1 have different effects on tumor vasculature, suggesting that targeting both pathways could have a more potent effect than targeting either pathway alone. We have created a bi-functional antibody conjugate targeting both ALK-1 and VEGF by conjugating VEGF-trap peptides to an anti-ALK1 monoclonal antibody. We demonstrate that our bi-functional molecule binds both ALK-1 and VEGF with high affinity in vitro, blocks both ALK-1 and VEGF pathways in HUVEC primary human endothelial cells and has good pharmacokinetics properties in mice. Finally, in a MDA-MB-231 xenograft model in SCID mice, the ALK-1/VEGF bi-functional antibody conjugate slowed tumor growth and prolonged survival with greater efficacy than anti-VEGF therapy alone.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2327. doi:1538-7445.AM2012-2327
Abstract 2331: Select epidithiodiketopiperazine (ETP) compounds disrupt the HIF-1α/p300 interaction and HIF-mediated transcription
Authors: Richardson Emily D. , Reece Kelie M. , Campbell Tessa J. , Pressler Heather , Pisle Stephen T. , Figg William D.
Published: August 2012

As solid tumors grow, regions in the tumor become hypoxic because of increased metabolism, heterogeneous and abnormal vasculature delivering oxygen within the tumor, and greater size. In order for the tumor to continue to grow, it depends on survival mechanisms to overcome this hypoxic state, including up-regulation in glycolysis, invasion and metastasis, and angiogenesis, or the growth of new blood vessels. The hypoxia-inducible factor (HIF) transcription factor complex is a major mediator of the cell's response to a hypoxic state and a tumor's survival mechanism under hypoxia. The complex is composed of the hypoxia-dependent HIF-1α subunit and a constitutively expressed HIF-1α subunit, and it requires the co-activator protein p300 to function. In the presence of oxygen, HIF-1α is rapidly degraded. Under hypoxia, HIF-1α forms a complex with HIF-1α and p300 in the nucleus to up-regulate transcription of pro-survival and angiogenic genes. Targeting this transcription factor complex in tumors could circumvent the tumor's hypoxia survival mechanism and lead to growth arrest and cell death. The compounds chetomin, chaetocin, and gliotoxin, members of the epidithiodiketopiperazine (ETP) family of fungal metabolites, have been shown to disrupt the HIF-1α/p300 interaction in an over-expression system. In the following experiments, we sought to validate the disruption of the HIF-1α /p300 transcription factor complex in cells, and to determine the associated downstream cellular effects. By co-immunoprecipitation of the complex from prostate cancer cells, we were able to show that treatment with those select ETPs under hypoxic conditions disrupted the HIF-1α/p300 complex. We performed an enzyme-linked immunosorbent assay (ELISA) to determine the effect of ETPs on levels of secreted vascular endothelial growth factor (VEGF), a major target of HIF transcription. We observed a decrease in VEGF concentration that correlated with ETP treatment under hypoxia. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to examine the transcription levels of other HIF target genes to validate that disruption of the HIF-1α/p300 complex by select ETP treatments indeed affects HIF-mediated transcription. In order to determine the antiangiogenic effects of these agents in vivo, ongoing animal studies in our lab are testing the efficacy of these ETPs in reducing tumor growth and determining feasibility of pre-clinical development. Targeting the HIF complex could circumvent a tumor's survival mechanism under hypoxia, and would be a new treatment strategy in solid tumors.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2331. doi:1538-7445.AM2012-2331
Abstract 2336: M402, a heparan sulfate mimetic, inhibits tumor revascularization and invasiveness after high-dose taxane treatment in a mouse breast cancer model
Authors: Schultes Birgit C. , Chu Chia L. , Long Alison , D'Alessandro Josephine , Honan Christopher M. , Kishimoto Takashi K. , Venkataraman Ganesh
Published: August 2012

Treatment with certain anti-cancer agents, particularly taxanes and sunitinib, can lead to mobilization of pro-angiogenic factors and an acute mobilization of endothelial progenitor cells (EPCs) and other stromal cells, which migrate to the viable tumor rim where they can enhance tumor vascularization, invasion and metastasis. This phenomenon has been linked to rapid tumor regrowth following chemotherapy or treatment with specific angiogenesis inhibitors and may thus diminish the long-term efficacy of the treatment. Stromal cells like EPCs are mobilized in response to circulating growth factors and chemokines (VEGFR, FGF, G-CSF, IL-6, SDF1α, etc.) that are induced by the drug or the progressing tumor. Many of these factors contain heparin binding domains for their anchorage to proteoglycans on cell surfaces or the extracellular matrix. We tested a novel heparan sulfate mimetic, M402, for its ability to inhibit EPC mobilization as well as tumor vascularization and invasion. Mice bearing orthotopic 4T1 breast carcinoma and treated with a single high dose of docetaxel (40 mg/kg) showed highly vascularized tumor rims by histology and Microfil perfusion followed by microCT (vessel volume of 5.8 ± 4.6 mm3 vs. 0.6 ± 0.3 mm3; vessel surface area of 153.9 ± 65.5 mm2 vs. 21.1 ± 6.7 mm2, respectively) as compared to the saline control group. The tumors also showed a higher rate of invasion through the peritoneal wall as compared to vehicle treated mice (5 of 16 mice vs. 2 of 16 mice, respectively). The effect correlated with mobilization of EPCs and other bone marrow derived stromal cells. Treatment with M402 did not show such a response but rather inhibited the effects of docetaxel when dosed concomitantly for 3-5 days, starting with the docetaxel administration. Analysis of the blood, tumor and bone marrow indicated that M402 prevented the mobilization of progenitor cells from the bone marrow and their recruitment into the tumor microenvironment, likely through disruption of the SDF-1α/CXCR4 axis. Furthermore, we observed synergistic anti-tumor and anti-metastatic activity of M402 with docetaxel in this 4T1 model. In conclusion, the experiments provide a rationale for the clinical investigation of M402 in combination with docetaxel or other agents that induce similar effects.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2336. doi:1538-7445.AM2012-2336