bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5074: Identification of Ras compartment-specific interacting proteins
Authors: Zheng Ze-Yi , Cheng Chiang-min , Fu Xin-Rong , Songyang Zhou , Chang Eric C.
Published: February 2011

Ras proteins are powerful molecular switches that control a wide range of functions, including cell growth, differentiation, apoptosis, cytoskeleton remodeling, etc. In humans, there are three RAS genes encode structurally highly similar Ras proteins that appear to have different functions in the cell. We have previously reported that a given Ras protein can signal from different cell compartments to control distinct functions in yeast. This present project aims to identify proteins that bind Ras in a cell compartment-specific manner in human cells to define how compartmentalized Ras signaling impacts tumor formation. We used the biomolecular fluorescence complementation (BiFC) method to screen a human cDNA library. Sixty two proteins were thus identified that bound the constitutively activated H-Ras (H-RasG12V), but not the GST control. These proteins also bound weakly to H-RasG12V whose effector binding motif has been mutated. To determine if these proteins can alter Ras functions, we first overexpressed them and then tested for their impacts on Ras-induced cell transformation and transcription. Only 25 of the isolated proteins could alter Ras signaling in these assays. Many of these 25 proteins are known to either associate with cellular membrane and/or regulate protein transportation, suggesting that as a group they highlight the importance of regulating Ras trafficking in the control of cell transformation. In particular, a number of isolated proteins may regulate endosome sorting, and we thus focused on two such proteins, CHMP6/VPS20 and VPS4A, for further investigation. We first validated the Ras binding by co-immunoprecipitation; furthermore the microscopy data showed that the binding is readily detectable in endosomes. Moreover, up- or down-regulating of level of either CHMP6/VPS20 or VPS4A dramatically inhibited Ras-induced transformation of NIH-3T3 cells. To determine how these molecules regulate Ras activities, we first examined whether they modulate Ras protein intracellular transport. Cell fractionation using sucrose gradients has shown that VPS4A knock-down could alter both wide-type and constitutively activated Ras membrane localization. In summary, we indentified Ras compartment-specific binding proteins by a cell-based approach. Many of the proteins are trafficking regulating proteins. In particular, two endosome sorting proteins CHMP6/VPS20 and VPS4A have been shown to regulate the oncogenic effects of Ras, possibly by localizing Ras proteins into proper cell compartments. We believe that the study of compartment-specific Ras interacting proteins is indispensable for better understanding the complex roles of Ras in oncogenesis of breast cancer as well as many other cancers.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5074.
Abstract 5076: Dissection of RAS downstream pathways in melanomagenesis
Authors: Mishra Prasun J. , Ha Linan , Rieker Jennifer , Sviderskaya Elena , Bennett Dorothy , Oberst Michael D. , Kelly Kathleen , Merlino Glenn
Published: February 2011

Cutaneous malignant melanoma is considered one of the most deadly human cancers, based upon both its penchant for metastatic spread and its typical resistance to currently available therapy. Long known to harbor oncogenic NRAS mutations, melanomas were more recently reported to be frequent bearers of activating mutations in BRAF, one of the effectors situated downstream of wildtype NRAS. RAS gene products (HRas, KRas and NRas) are 21kd G-proteins that serve as molecular switches converting cell-surface kinase activation events to nuclear events, thus influencing cell behavior. The major downstream effectors of Ras are the Rafs (ARaf, BRaf and CRaf), phosphatidylinositol 3-kinase (PI3K) and the Ral guanine exchange factors (RalGEF). NRAS and BRAF mutations are rarely found in the same melanoma, suggesting that they may possess important overlapping oncogenic activities. Here we compare and contrast the oncogenic roles of the three major NRas downstream effectors, Raf, PI3K and RalGEF, using genetically engineered Arf-deficient immortalized mouse melanocytes as a model system. Activation of each of the three major downstream pathways of NRas has overt phenotypic effects on immortalized mouse melanocytes, many distinct. Our data indicate a prominent role for BRaf and PI3K in melanocyte senescence and invasiveness, respectively. More surprisingly, we found that constitutively activated RalGEF stimulated melanocyte invasiveness and anchorage-independent growth almost as impressively as NRasQ61K. The suppressive effects of expression of a dominant negative Ral corroborated the role of the RalGEF-Ral pathway in anchorage-independent growth. Although no single downstream pathway could recapitulate all of the consequences of oncogenic NRas expression, melanocytes expressing constitutively activated mutants of all three NRas downstream factors (RLF-CAAX plus p110α-CAAX plus BRafV600E) achieved soft agar colony forming efficiency that was fully comparable to those expressing NRasQ61K alone. In conclusion, our data suggested a distinct function for all three NRas downstream pathways - Raf, PI3K and Ral in melanocyte senescence, invasiveness and transformation, respectively. Importantly, our finding that constitutive RalGEF activation confers malignant phenotypes in melanocytes, particularly anchorage-independent growth, indicates that this often overlooked pathway should be more carefully evaluated as a possible therapeutic target.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5076.
Abstract 5077: Signaling through the cytoplasmic tail of MUC1 directly activates transcription of the metastasis promoting growth factor CTGF in pancreatic adenocarcinoma
Authors: Behrens Michelle , Grandgenett Paul , Bailey Jennifer , Singh Pankaj , Yi Chun-Hui , Hollingsworth Michael
Published: February 2011

Transmembrane mucin MUC1 is highly expressed in pancreatic adenocarcinoma and contributes to tumor growth, invasion, and metastasis. Differential phosphorylation of the cytoplasmic tail of MUC1 (MUC1.CT) by growth factor receptor tyrosine kinases is associated with morphogenetic signal transduction, as it enables interactions with signal transducers and leads to nuclear accumulation of MUC1.CT. MUC1.CT contains no known DNA binding motif but localizes to promoters of genes implicated in tumorigenesis in complex with transcription factors, modulating their regulatory activity. Using microarray analysis, we identified genes differentially transcribed in S2013 pancreatic adenocarcinoma cells stably overexpressing MUC1. These data were correlated with transcriptional targets of MUC1.CT identified on a genome-wide scale in pancreatic adenocarcinoma cells using a combination of chromatin immunoprecipitation and promoter array methodologies (ChIP-on-chip). We further investigated the transcriptional regulation of one novel target, connective tissue growth factor (CTGF/CCN2), by MUC1.CT. CTGF is highly expressed in pancreatic cancer and is a known mediator of tumor growth, angiogenesis, and metastasis. Overexpression of MUC1 significantly increased CTGF expression in pancreatic tumor cells in response to EGF, PDGF-BB, or HGF stimulation. The MUC1.CT becomes phosphorylated by EGFR, PDGFR-beta, and c-Met and overexpression of MUC1 constructs harboring mutations within the target phosphorylation motifs for these receptor tyrosine kinases reduced CTGF expression. We determined that MUC1.CT directly regulated the CTGF gene transcription through co-occupancy with transcription factors at several regions within and surrounding the CTGF promoter. MUC1 overexpression promoted localization of mutant p53 (R273H) and beta-catenin to the CTGF promoter, while decreasing occupancy by a repressor of CTGF transcription, c-Jun. We propose that MUC1, when overexpressed in pancreatic adenocarcinoma, enables expression of CTGF in response to growth factor stimulations by promoting mutant p53 and beta-catenin transcriptional activity and alleviating CTGF transcriptional repression by c-Jun. Taken together, these results reveal a novel signaling pathway that selectively induces CTGF expression in MUC1-overexpressing pancreatic tumor cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5077.
Abstract 5086: Pemetrexed induces a strong activation of AMPK in human carcinoma cells, even in the absence of LKB1
Authors: Rothbart Scott B. , Racanelli Alexandra C. , Moran Richard G.
Published: February 2011

AMP-activated protein kinase (AMPK) is a major cellular energy sensor, functioning as a regulator of signaling cascades controlling protein, lipid, and carbohydrate metabolism. Because of the dependence of many tumors on the deregulation of the PI3K-AKT-mTOR signaling cascade, the discovery of AMPK activators is an attractive approach for cancer therapeutics, aimed at reinstating control over cellular metabolic functions altered during carcinogenesis rather than inhibiting cellular targets. The antifolate pemetrexed is FDA approved for first line treatment of mesothelioma and non-small cell lung cancer (NSCLC). While pemetrexed acts primarily as a thymidylate synthase inhibitor, we discovered that the drug is also an inhibitor of aminoimidazolecarboxamide ribonucleotide formyltransferase (AICART), the second folate-dependent enzyme of de novo purine synthesis. The inhibition of AICART by pemetrexed resulted in a marked accumulation of the reaction substrate, ZMP, a known allosteric activator of AMPK in several human carcinoma cell lines. The mammalian target of rapamycin complex 1 (mTORC1) was inhibited following pemetrexed treatment, as determined by hypophosphorylation of S6K1 and 4EBP1, and this effect was dependent on AMPK activation. Co-administration of GSK690693, a pan AKT inhibitor prolonged mTORC1 inhibition following pemetrexed treatment and offset the consequences of negative feedback activation of mTORC1 through PI3-kinase and mTORC2 activity. Interestingly, cells insensitive to mTORC1 inhibition following AMPK activation retained sensitivity to the secondary effect of pemetrexed, apparently due to inhibition of lipid synthesis through phosphorylation of acetyl-CoA carboxylase by AMPK. We concluded that the growth inhibitory effect of pemetrexed following AMPK activation in human carcinoma cells was due to a combined effect of inhibition of mTORC1 signaling and lipogenesis, both of which were mediated by activation of AMPK. Somewhat surprisingly, several cell lines null for LKB1, the major upstream kinase of AMPK, retained sensitivity to the secondary effects of pemetrexed mediated through AMPK activation. This suggests that the effect of pemetrexed on AMPK activity will be preserved in tumors lacking LKB1, a common genotype of human NSCLC. Hence, pemetrexed exerts its effects on carcinoma cells by both inhibition of traditional antifolate targets and by a novel mechanism, activation of AMPK. (Supported in part by NIH Grant CA-27605)

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5086.
Abstract 5088: In vitro induction of a more aggressive prostate cancer phenotype by exposure to obese sera
Authors: Salcedo Ramona , Cavazos David , Hursting Stephen , Angel Rebecca De , deGraffenried Linda , Beeram Muralidhar
Published: February 2011

Prostate cancer continues to be the second-leading cause of cancer related deaths for men in the United States. Obesity is a known risk factor for the progression of prostate cancer. Studies have shown an association between obesity and chronic inflammation. Inflammation may contribute to prostate growth by promoting benign prostatic hyperplasia or inducing neoplastic changes. The mechanism(s) by which obesity promotes an aggressive prostate cancer is not fully understood. Investigating these mechanisms has been hampered by the lack of appropriate in vitro and in vivo model systems. Our main hypothesis is that obesity-induced chronic inflammation promotes metastatic prostate cancer through modulation of key signaling pathways, including NF-[[Unable to Display Character: ĸ]]B. Our first objective was to develop an appropriate in vitro model system to recapitulate the molecular events associated with obesity. In this current study, we obtained sera from obese and non-obese mice. Using an adipokine protein array 3 independent profiles were identified in obese mice sera that are consistent with metabolic obesity: metabolic syndrome, pro-metastatic and pro-inflammatory profile. We treated LNCaP prostate cancer cells with sera from obese and non-obese mice to determine physiological responses associated with an aggressive phenotype. We observed obese sera promoting invasion and increasing cell motility while having no effect on proliferation. In addition, obese mouse sera induced an EMT phenotype, evidenced by cytoplasmic dispersion of e-cadherin from cell-cell junctions and an increase in vimentin, which corresponded in greater tumor invasion. This study suggests that we can use an in vitro system to mimic the physiological changes in tumor biology induced by exposure to obese sera to investigate the molecular changes that induce a more aggressive phenotype.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5088.
Abstract 5093: Pseudomonas aeruginosa azurin secretion is stimulated by aldolase A from human malignant melanoma cells
Authors: Choi Juliana K. , Gupta Tapas K. Das
Published: February 2011

Recent reports have shown that Pseudomonas aeruginosa secrete increased levels of a redox protein, azurin, when co-cultured with human cancer cells. Previous data have demonstrated that azurin preferentially enters and induces cytotoxicity in several cancer cell lines; this same entry and cytotoxicity were not observed with normal cells. In the present study, P. aeruginosa, human malignant melanoma cells (Mel-2), and non-malignant melanocytes (CMN) were used as a model system to determine how P. aeruginosa distinguishes a cancer cell from a normal cell. P. aeruginosa were directly co-incubated with Mel-2 and CMN, and Western blot analyses were used to analyze levels of azurin secretion. A dose-dependent increase in azurin secretion was demonstrated upon co-incubation of P. aeruginosa and Mel-2, and azurin secretion was increased greater than 10-fold in the presence of Mel-2 compared to CMN. Also, real-time PCR assessment of azurin transcription demonstrated a 2-fold increase in azurin transcripts when P. aeruginosa were co-incubated with Mel-2. Next, a Transwell system was used in which P. aeruginosa and Mel-2 were co-incubated to allow secretion exchange but prevent physical contact. Using this system, it was demonstrated that azurin release is stimulated by soluble extracellular factors secreted by Mel-2 but not CMN. Using Coomassie staining and mass spectrometry techniques, aldolase A, a glycolytic enzyme upregulated in many cancer cells, was identified as a stimulating factor for azurin secretion. Cancer and normal cell line pairs of varying histogenesis were examined for basal aldolase A levels, and aldolase A was generally found to be elevated in cancer cell lines compared to their normal counterparts. These same cell line pairs were co-incubated with P. aeruginosa and azurin secretion directly correlated with aldolase A levels. The results suggest that secretion of azurin is, at least in part, mediated by aldolase A. In close proximity, P. aeruginosa may detect aldolase A in cancer cells and secrete azurin as a defense mechanism to induce cytotoxicity in Mel-2. Using P. aeruginosa, Mel-2 cells, and CMN cells as a model system, it was demonstrated that inherent differences between cancer cells and normal cells can be detected by bacteria and subsequently lead to a preferential interaction.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5093.
Abstract 5118: Rho/ROCK pathway mediates plasticity of scirrhous gastric carcinoma cell motility
Authors: Tasuku Matsuoka , Yashiro Masakazu , Kato Yukihiro , Shinto Osamu , Onoda Naoyoshi , Sawada Tetsuji , Nakata Bunzo , Ohira Masaichi , Hirakawa Kosei
Published: February 2011

The small guanosine triphosphatase (GTPase) Rho and its downstream effector Rho-associated kinase (ROCK) is one of a key mediator involved in controlling focal adhesions and the dynamics of actin stress fibers. This pathway has been shown to play important roles in the regulation of various cellular processes, such as cell shape changes and adhesion dynamics that drive migration. The underlying molecular mechanisms for the function of Rho/ROCK pathway leading to the development and progression in scirrhous gastric carcinoma cells have not been defined. We investigated the role of the Rho/ROCK pathway in the adhesion and invasion of gastric carcinoma cells, using a human scirrhous gastric cancer cell line, OCUM-2MD3. ROCK inhibitor, Y27632 significantly increased the adhesion of OCUM-2MD3 cells to the extracellular metrix (ECM) protein matrigel, but had no effect on attachment to polylysine. Further examination using ECM components showed enhanced binding ability was obtained only in laminin, but not in fibronectin, Type I collagen, and Type IV collagen. Reverse transcriptional (RT)-PCR analysis revealed that integrin subunits α3-integrin was clearly up-regulated by treatment with Y-27632 in OCUM-2MD3 cells, reflecting the specificity of ECM protein showing high adhesiveness property of gastric carcinoma cells. ROCK inhibitor also enhanced the invasion of OCUM-2MD3 cells through matrigel. The fact that Y27632 up-regulated the expression of membrane-type 1 matrix metalloproteinase by RT-PCR suggested the possibility that increased invasion ability of OCUM-2MD3 cells was related to proteolytic activity of extracellular matrices. Time-lapse microscopy showed conversion of OCUM-2MD3 cells from elongated to more round morphology with bleb in the presence of Y27632, suggesting that inhibition of Rho/ROCK pathway undergo a so-called ‘amoeboid to mesenchymal’ transition. Taken together, these data suggests that Rho/ROCK regulate plasticity of metastatic gastric carcinoma via mesenchymal-amoeboid transition, leading to clarify the specific progression of scirrhous gastric carcinoma.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5118.
Abstract 5121: Laminin 511 on human gastric carcinoma cells is a ligand for hepatic asialoglycoprotein receptor involved in liver metastasis
Authors: Ueno Suguru , Nodera Yoshiaki , Gomez Laura Santos , Higashi Nobuaki , Irimura Tatsuro
Published: February 2011

– Background and Aim – Gastric cancer often metastasizes to the liver and galactose-type lectins were previously proposed to play an important role in hepatic metastases because intravenous administration of galactose at the time of surgical removal of gastric cancer was reported to extend the survival of the patients. Asialoglycoprotein receptor (Asgpr) is a galactose-type calcium-type lectin and a primary candidate molecule involved in this process. Variant cell lines of human gastric carcinoma, AZ521 cells, were obtained by sorting for high and low levels of cell surface bindings of recombinant Asgpr and designated as AZhigh and AZlow cells, respectively. The present report is to assess the metastatic potential of these cell lines and determine the ligand for Asgpr on the surfaces of these gastric carcinoma cells.

– Materials and Methods – AZhigh and AZlow cells were injected into nude mice via portal vein and metastatic growth in the liver were evaluated 14 days later. AZhigh and AZlow cells were biotinylated at the cell surfaces and the lysates were prepared. Components bound to immobilized Asgpr were collected and analyzed by SDS-PAGE then differentially expressed components were subjected to MALDI-TOF MS analysis after trypsin digestion. Specific monoclonal antibodies specific for particular components such as laminin α5 chain were used to confirm the identified proteins from the carcinoma cells.

– Results and Discussion – Metastatic growth in the liver was more prominent with AZhigh cells than AZlow cells. AZhigh cells adhered to immobilized Asgpr more rapidly than AZlow cells. From components eluted from immobilized Asgpr, laminin α5, β1, and γ1 chains were identified. By flow cytometric analysis, anti-human laminin α5 antibody bound to AZhigh cells but the binding to AZlow were negligible. Similar difference was observed by immunoprecipitation. Laminin α5 chain was also in the culture supernatants of AZhigh cells, but not those of AZlow cells. These results suggest that laminin 511 differentially expressed between AZhigh and AZlow cells and acted as the ligands for Asgpr. Further study focusing on laminin 511 will be needed for elucidating the mechanism of hepatic metastases of gastric cancer.

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

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5121.
Abstract 5125: The identification of Gelsolin as a potential binding partner of Nm23
Authors: Marino Natascia , Marshall Jean-Claude A. , Collins Joshua , Zhou Ming , Veenstra Timothy , Steeg Patricia
Published: February 2011

Nm23 was the first metastasis suppressor gene to be identified. To better understand the biological mechanism leading to its anti-metastatic properties, we have investigated new potential binding-proteins of Nm23. Using Mass-spectrometry, we analyzed proteins obtained with co-immunoprecipitation of Nm23 in a mouse mammary cancer cell line overexpressing the human and murine forms of Nm23 (4T1-Nm23H1-flag and 4T1-Nm23M1-flag). Co-immunoprecipitations were also performed from primary tumors and liver metastases obtained from injection of these cell lines into Balb/C mice. Several proteins were found to bind Nm23, both in vitro and in vivo specimens. In addition to previously published Nm23 interacting proteins, we identified new binding partners including the actin-binding protein Gelsolin.

Using immunoprecipitation assays, the interaction between Gelsolin and Nm23 was validated in a murine cell line (4T1- Nm23H1/M1 clones) and then in human breast carcinoma cell lines with low (MCF-7) and high metastatic potentials (MDA-MB-435, MDA-MB-231). In the highly metastatic MDA-MB-231 cell line, the interaction was observed only after the overexpression of Nm23 in the system. These results suggest that a Nm23-gelsolin interaction may only take place when relatively high levels of Nm23 are expressed.

Moreover, we observed that this interaction involves not only the native form of Gelsolin but also Gelsolin-fragments, NH2-terminal and COOH-terminal, which are generated by the action of caspase-3 cleavage. Full-length Gelsolin binds and severs actin polymers in a Ca2+-dependent manner, whereas caspase-3-cleaved Gelsolin severs actin polymers independent of Ca2+. The overexpression of the NH2-terminal fragment has been shown to induce apoptosis, whereas the COOH Terminal fragment has been shown to have an anti-apoptotic effect. We are therefore interested in exploring which Gelsolin-fragment may be specifically binding to Nm23, in order to determine the biological function regulated by this interaction. Our results show a novel Nm23 binding partner, Gelsolin, which is involved in the actin-cytoskeleton regulation and may, in part, explain the motility inhibitory activity of Nm23. The exact interactions between Nm23 and Gelsolin are currently being explored. In particular we are evaluating the role of this interaction in the anti-metastatic effects of Nm23, both human and murine.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5125.
Abstract 5128: Identification of RhoGEFs within the TWEAK-R (Fn14)-Rac1 signaling pathway that mediate glioblastoma cell invasion
Authors: Ennis Matthew J. , McDonough Wendy S. , Chan Amanda , Symons Marc H. , Winkles Jeffrey A. , Salhia Bodour , Loftus Joseph C. , Tran Nhan L.
Published: February 2011

Glioblastoma multiforme (GBM) is the most malignant primary adult brain tumor. Despite efforts at surgical resection of the glioma mass, invasive cells are always left behind and the tumor will inevitably recur and kill the patient. As such, novel therapeutics targeting pro-invasive factors could improve neurological outcomes and survival for these patients. This requires a detailed understanding of the mechanisms driving glioma migration and invasion. We have been investigating whether the multifunctional cytokine tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and its receptor fibroblast growth factor-inducible 14 (Fn14) regulate glioma cell invasive activity. We have previously demonstrated that Fn14 expression was elevated in the invasive rim of GBM specimens and migrating glioma cells in vitro. The Fn14 signaling axis is known to drive glioma invasion via Rac1. We have previously reported that Ect2, a guanine nucleotide exchange factor (GEF) for Rho family GTPases, including Rac1 and Cdc42, is overexpressed in GBM, and that overexpression of Ect2 correlates directly with tumor grade and inversely with patient survival. In this study we show that Ect2 regulates Rac1 activation downstream of Fn14. Depletion of Ect2 by siRNA duplexes abrogates Fn14-induced Rac1 activation and subsequent glioma cell migration and invasion. We also found that Cdc42 activation by TWEAK is directly mediated by Ect2. Interestingly, depletion of Cdc42 expression impairs TWEAK-induced Rac1 activation and also results in a significant reduction of glioma cell migration and invasion. This suggests that Cdc42 is, in part, important for Rac1 activation downstream of the TWEAK-Fn14 signaling pathway and argues that another GEF(s) may be involved in the Fn14-Rac1 signaling axis. Recently, we have identified Trio as an additional GEF that activates Rac1 downstream of Cdc42. It is also known that Trio expression correlates directly with brain tumor grade and inversely with patient survival. siRNA-mediated depletion of Trio inhibits TWEAK-induced Rac1 activation but not TWEAK-induced Cdc42 activation. Therefore, delineating the mechanisms of Fn14-RhoGEF-RhoGTPase signaling pathway, may lead to identification of novel targets that can serve as possible points of therapeutic intervention. (Supported by NIH R01-CA130940)

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5128.
Abstract 5130: Src and Caveolin-1 reciprocally regulate metastasis via Rho Kinase
Authors: Thomas Shibu , Overdevest Jonathan B. , Nitz Mathew , Williams Paul , Schwartz Martin A. , Theodorescu Dan
Published: February 2011

In human bladder cancer, overexpression of Caveolin-1 (Cav-1) and downregulation of Src expression and kinase activity correlate with aggressive behavior suggesting they may have causal and perhaps interactive roles in this process. Here we show that Cav-1 and Src are reciprocally expressed in the same tumors, suggesting they act on a similar pathway. RNAi mediated Cav-1 depletion or expression of constitutively active Src in invasive human urothelial cancer cells UMUC-3 and KU-7 resulted in loss of stress fibers and reduced capacity to migrate. Cav-1 depletion was associated with loss of pY14 Cav-1 while increased p190RhoGAP was seen with Src over-expression while both led to a significant reduction in RhoA and RhoC levels and activity. Conversely, expression of Cav-1 but not the non-phosphorylatable mutant of Cav-1 or suppression of Src using PP2 or Src RNAi in noninvasive human urothelial RT4 cancer cells, increased RhoA and RhoC GTP and cell migration. Use of ROCK inhibitor Y27632 in RT4 cells completely abolished enhanced migration induced by Cav-1 expression or that associated with Src inhibition. Furthermore, metastatic lung colonization of UMUC-3 cells expressing constitutively active Src or depleted of Cav-1 was significantly reduced while having marginal effect on subcutaneous tumor growth. Moreover, ROCK inhibitor prevented bladder cancer lung metastasis in vivo. Our findings indicate Src is metastasis suppressor protein in bladder cancer and describe a novel relationship between Cav-1 and Src in regulating RhoA and RhoC providing a unifying molecular mechanism for prior descriptive studies suggesting a role for these proteins in bladder cancer progression.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5130.
Abstract 5132: 14-3-3sigma coordinates breast tumor invasion by regulating cytoskeletal dynamics
Authors: Boudreau Aaron , Geyer Felipe C. , Wang Daojing , Reis-Filho Jorge S. , Bissell Mina J.
Published: February 2011

14-3-3sigma was initially described as a tumor suppressor silenced in about 80% of breast tumors. Surprisingly however, in two independent culture models of breast cancer progression, we discovered 14-3-3sigma expression increased as cells transition towards malignancy, and that it contributed to motility and tumor invasion into surrounding tissues in vivo. Immunohistochemical analysis showed that 14-3-3sigma expression preferentially identified a subset of invasive breast carcinomas, and in independent breast cancer patient cohorts, 14-3-3sigma correlated with poor clinical outcome. We observed several lines of evidence that 14-3-3sigma regulates cytoskeletal architecture in cells, providing a molecular mechanism by which 14-3-3sigma may coordinate tumor progression and invasion.

Funding is provided by a DOD BCRP Predoctoral Fellowship and a CBCRP Dissertation Award to AB, as well as DOE, NCI, NIH, and DOD grants to MJB.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5132.
Abstract 5134: Mechanistic dissection of EWS-FLI impact on Ewing's sarcoma cell behavior
Authors: Chaturvedi Aashi , Lessnick Stephen L. , Beckerle Mary C.
Published: February 2011

Ewing's sarcoma is caused by the oncogenic fusion of the EWS and FLI genes to encode a chimeric EWS-FLI protein product. The disease is often metastatic at presentation. This study aims to define the molecular mechanism by which the EWS-FLI oncoprotein influences metastatic potential by testing the impact of EWS-FLI expression on cytoarchitecture, adhesion, and migration of Ewing's sarcoma cells. We compared the behavior of A673 Ewing's sarcoma cells with A673 cells in which we knocked down expression of EWS-FLI by RNA-interference. Knock-down of EWS-FLI results in enhanced cell spreading and the establishment of robust actin stress fibers and focal adhesions. Consistent with these morphological attributes, a quantitative cell adhesion assay revealed that parental A673 cells are less adhesive than those in which EWS-FLI expression was reduced by RNA interference. Knockdown of EWS-FLI in A673 cells is also associated with increased cell motility and loss of anchorage-independent cell growth. Gene expression profiling revealed that expression of EWS-FLI causes downregulation of transcripts encoding several focal adhesion proteins and integrin adhesion receptors by 2-4 fold. By Western immunoblot analysis, we demonstrated that knock-down of EWS/FLI oncoprotein expression in human A673 cells leads to increased expression of focal adhesion constituents such as the actin regulator, zyxin. Introduction of a zyxin expression construct into A673 Ewing's sarcoma cells was sufficient to restore the integrity of the actin cytoskeleton and focal adhesions, and to enhance cell adhesion. In contrast, restoration of zyxin expression in A673 sarcoma cells did not affect cell motility, matrigel invasion, or anchorage-independent cell growth. Thus, oncogenic transformation by EWS-FLI is associated with changes in cytoskeletal architecture, cell adhesion, cell motility/invastion, and cell growth control. Re-expression of a single focal adhesion protein, zyxin, is sufficient to restore cytoskeletal integrity and partially rescue the adhesion deficits seen in A673 sarcoma cells. These studies provide evidence for a zyxin-dependent pathway downstream of EWS-FLI that controls cell morphology and adhesion, as well as zyxin-independent pathways that influence motility/invasion and anchorage-independent cell growth. Loss of adhesion coupled to reduced zyxin expression may be an early step in the establishment of metastatic potential of Ewing's sarcoma cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5134.
Abstract 5136: Sulfiredoxin promotes cell migration through direct interaction with non muscle myosin IIa
Authors: Bowers Robert , Townsend Danyelle M. , Manevich Yefim , Tew Kenneth D.
Published: February 2011

Redox homeostasis is frequently dysregulated in cancer cells. In particular, glutathione pathways can be imbalanced and impact cell proliferation and death pathways. S-glutathionylation is the addition of glutathione to basic cysteine residues in certain proteins producing significant changes in their structure/function. Because this post-translational modification is dynamic and reversible, the S-glutathionylation cycle can provide the framework for important cell signaling events in cancer cells. We have shown that sulfiredoxin (Srx) plays a role in deglutathionylation and interacts with proteins such as the phosphatase PTP1B, actin and peroxiredoxin 6 (Prdx 6). Redox regulation of actin modulates cell adhesion and focal contacts. In the present study, we examined the role of Srx in cell migration, invasion and potentially metastasis. Srx was over-expressed by transfection in lung cancer cells (A549). Phalloidin staining showed that Srx over-expression (A549-Srx) led to phenotypic changes in cell adhesion through altered focal adhesion contact points and the cells were more refractory to trypsin release. Relative to wild type, A549-Srx cells showed enhanced migration and invasion in the scratch- and Boyden chamber assays. Proteomic analysis through mass spectrometry demonstrated that Srx interacts with non-muscle myosin IIa (NMIIa) in A549 cells. Reciprocal co-immunoprecipitation of Srx and NMIIa validated this interaction under non-stressed conditions. Either through protein: protein interaction or through redox regulation (or both), Srx appears to be an important mediator of the cell motility and invasive properties of the A549 human lung cancer cell line.

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

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5136.
Abstract 514: Integrins dependent regulation of endothelial cell migration and tube formation by α6(IV)NC1
Authors: Akulapalli Sudhakar , Boosani Chandra S.
Published: February 2011

Peptides or short protein domains derived from extracellular matrix were shown to possess anti-tumor properties. One such molecule, α6(IV)NC1 was derived from the carboxy terminal non-collagenous domain fragment of α6 chain, type (IV) collagen and was found to inhibit angiogenesis and tumor growth but the mechanism by which it inhibits the growth of solid tumors has not been reported yet. In the present study we demonstrate that the biological functions of α6(IV)NC1 are attributed to its binding to different integrins. In addition, we also demonstrate that α6(IV)NC1 binds to α3β1and α5β1 integrins and competes with the cells binding to α1β1 integrins on type IV collagen, and / with α5β1 integrins on fibronectin and inhibits migration and tube formation of human vascular and lymphatic endothelial cells. We further evaluated the role of α3β1 and α5β1 integrins in tube formation and regulation of cell migration by α6(IV)NC1. While both the integrins (α3β1 and α5β1) are required for the inhibition of tube formation by α6(IV)NC1 in human vascular and lymphatic endothelial cells, only α3β1 integrin was found to regulate lymphatic endothelial cells migration. In addition we also demonstrate that α6(IV)NC1 inhibits tumor growth, tumor lymphatic angiogenesis and circulating endothelial cells in-vivo. These in-vitro and in-vivo findings identify that α3β1 and α5β1 integrins are critical for α6(IV)NC1 mediated inhibition of angiogenic and lymph angiogenic signaling and inhibition of tumor progression. Collectively, our findings first time demonstrate that α6(IV)NC1 is a potent therapeutic candidate for targeting tumor angiogenesis and tumor lymphatic angiogenesis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 514.
Abstract 515: Interaction of CD44 with matrix metalloproteinase-9 (MMP-9) is crucial to maintain adhesion, migration and invasion in glioblastoma xenograft cells
Authors: Vanamala Sravan K. , Gondi Christopher S. , Veeravalli Krishna K. , Chetty Chandramu , Fassett Daniel , Klopfenstein Jeffrey D. , Dinh Dzung H. , Gujrati Meena , Rao Jasti S.
Published: February 2011

CD44, a transmembrane receptor for hyaluronic acid, is comprised of an extracellular domain (ECD) and intracellular domain (ICD). CD44 is implicated in various adhesion-dependent cellular processes, including cell migration, tumor cell metastasis and invasion. Here, we have generated constructs for knockdown of CD44 corresponding to ECD and ICD as well as to selectively overexpress either ECD or ICD. CD44 knockdown resulted in a more than 50% reduction in cellular adhesion, migration, and invasion as determined by MTT, wound closure and Matrigel assays in glioblastoma xenograft cell lines (4910 and 5310). Zymography analysis of conditioned medium from CD44 knocked down 4910 and 5310 cells showed decreased gelatinolytic activity of MMP-9; however, western blot analysis of cell lysates did not show any change in MMP-9 expression. Based on these results, we sought to further determine the relationship of CD44 with MMP-9, which is known to play an important role in cellular adhesion, migration and invasion. Immunoprecipitation analysis of cell lysates from 4910 and 5310 cells revealed an interaction between CD44 and MMP-9. Of note, siRNA-mediated knockdown of MMP-9 resulted in inhibition of CD44 cleavage. Selective overexpression of ECD or ICD with downregulation of endogenous CD44 showed that ECD expression was able to recover cellular adhesion and gelatinolytic activity of MMP-9. In contrast, as seen in the MTT assay results, ICD overexpression recovered cell growth but not adhesion or MMP-9 gelatinolytic activity. Taken together, our results suggest the interaction between CD44 and MMP-9 plays an important role in maintaining cellular growth, adhesion, invasion and migration in the glioblastoma xenograft cell lines 4910 and 5310.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 515.
Abstract 517: Expression of PTRF/cavin-1 in prostate cancer cells reduces migration via decreased MMP9 expression
Authors: Aung Cho S. , Hill Michelle , Parton Robert , PARAT Marie-Odile
Published: February 2011

Caveolin-1 expression and secretion are increased in metastatic prostate cancer and caveolin-1 seems to contribute to prostate cancer growth and metastasis. Caveolin-1 was long believed to be sufficient for caveolae formation, but a cytoplasmic protein named PTRF (Polymerase I and Transcript Release Factor) or cavin-1 was recently found to be required, in concert with caveolin-1, for the formation and functions of caveolae. We hypothesized that PTRF expression could modulate prostate cancer cell migration. In metastatic PC3 prostate cancer cells that express abundant caveolin-1 but no PTRF, heterologous PTRF expression restores caveolae formation and caveolin-1 distribution. We show that PTRF-expressing PC3 cells exhibit decreased migration, and that this effect is mediated by reduced matrix metalloprotease-9 (MMP9) production. We further show that MMP9 production is affected at the transcriptional level, and independently from caveolae formation. Of all the cavin family members, PTRF-cavin-1 is the only one to dramatically reduce MMP9 expression. Taken together, our results suggest that PTRF-cavin-1 expression alters prostate cancer aggressiveness.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 517.
Abstract 5175: L1 stimulation of human glioma cell motility correlates with FAK activation
Authors: Yang Muhua , Li Yupei , Boulos Magdy I. , Galileo Deni S.
Published: February 2011

Our previous studies showed the expression and proteolysis of neural adhesion molecule L1 (CD171) in human glioma cell lines and human glioma surgical samples. The proteolysis of this molecule was suggested to autocrine stimulate glioma cell motility through binding to integrin receptors (Yang et al., 2009). To further investigate the mechanism of this stimulation, in this study, a lentivirally introduced shRNA was used to attenuate L1 expression in human glioma T98G cells (T98G/shL1 cells), which resulted in 95% decreased expression compared to empty vector infected control cells. L1 attenuated cells exhibited 50% decreased cell motility compared to control cells as shown by time lapse microscopy and the quantitative “Super Scratch” assay. These slower T98G/shL1 cells showed a 68% decrease in FAK activity and an approximately 36% increase in focal complex size compared to control cells. These results suggest that T98G/shL1 cells had decreased focal complex turnover, which resulted in their slower migration. Conditioned media containing the L1 ectodomain was made from CHO cells infected with a lentiviral vector expressing the human L1 ectodomain sequence. Adding L1 conditioned media 1) rescued the cell motility in T98G/shL1 cells and 2) increased cell motility in U118 cells (a grade III human glioma cell line lacking of endogenous L1 expression) by 30% compared to the control cells. These results support a model for stimulation of glioma cell motility through the L1 ectodomain after ADAM10 proteolysis (Yang et al., 2009). In order to examine L1 function in glioma cell invasion into brain tissue, a novel embryonic chick model was used. Injection of GFP labeled T98G cells and dissociated cells from human glioma surgical samples into embryonic chick brains resulted in extensive invasion after 4 days, and this in vivo system is being used to study L1 function in glioma cell invasion. Confocal microscopy of immunostained tissue sections revealed that cell surface L1 was missing in cells located at the margin of the tumor as opposed to cells in the tumor mass. These results are consistent with our previous finding that L1 was expressed on the surfaces of confluent T98G cells in culture, but was lost from the cell surface along the edge of a scratch into which cells migrated. These studies suggest a general mechanism where the L1 ectodomain autocrine stimulates glioma cell motility and invasion by binding to integrin receptors, which activates FAK and thereby increases turnover of focal complexes. Supported by NIH INBRE.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5175.