bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5158: Differentiation of glioblastoma stem-like cells induced by retinoic acid
Authors: Ying Mingyao , Sang YingYing , Lal Bachchu , Sun Peng , Laterra John , Xia Shuli
Published: February 2011

We investigated the molecular and cellular responses of glioblastoma stem-like cells (GBM-SCs) to all-trans retinoic acid (RA)-induced differentiation. Four GBM-SC lines were used. Under neurosphere culture conditions, GBM-SCs grow as non-adherent neurospheres. RA (10 μmol/l) treatment for 48 h induced cells to adhere to culture surfaces and generate processes. RA inhibited G1/G0 to S transition, decreased cyclin D1 expression (50%) and increased p27 expression (2-fold). Immunofluorescence staining, western blot and flow cytometry analyses showed that RA induced the expression of differentiation markers Tuj1 and GFAP and inhibited nestin expression. RA treatment reduced the number of cells expressing CD133 from 44% to 10%. We measured the effects of RA on neurosphere number and size. RA treatment dramatically decreased (∼90%) the number of neurospheres > 50 um diameter. Clonogenic assay showed that RA reduced the colony formation ability of GBM-SCs in soft agar. Microarray analysis was performed to determine the global gene expression profile of RA-induced GBM-SC differentiation. Data were analyzed using Significance Analysis of Microarrays (SAM) and the false discovery rate was set at 20%. Approximately 350 genes were altered after RA treatment for 48h; ∼20% were down-regulated and 80% were up-regulated. We selected 18 candidates and validated with real-time PCR. All 18 candidates exhibited the same pattern of up- or down-regulation by RA. Western blot analysis for some candidate gene products also confirmed the alteration by RA at the protein level. A web-based (portal.genego.com) molecular pathway analysis of RA-induced gene expression profiling identified several pathways altered during RA-induced differentiation process. The most prominent of them was Notch signaling. Notch pathway members Delta-like 1 (DLL1), Notch 1, NCID, NEXT and Notch 1 precursor were found to be down-regulated by RA. Only Delta-like 4 (DLL4) was up-regulated by RA. We also found that cyclin D, cyclin D1, PP2A and SMAD3, gene products involved in G1-S transition, were down-regulated by RA. This is consistence with our findings that RA induces cell cycle arrest. Numerous mechanisms involved in cell adhesion, cell-matrix interaction and cytoskeleton remodeling were also affected by RA. This analysis provides guidelines to further explore pathways and mechanisms during RA-induced GBM-SC differentiation. Our findings may be applicable to identify new molecular target applicable to differentiation therapies for glioblastomas. This will also stimulate the development of GBM stem cell based pre-clinical experimental models with the potential to improve mechanistic and pre-clinical therapeutic research.

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 5158.
Abstract 5163: Does melanoma metastasis recapitulate an embryonic neural crest invasion program
Authors: Bailey Caleb M. , Ythier Sylvain , Kulesa Paul M.
Published: February 2011

Melanocytes derive from a highly invasive and pluripotent embryonic cell population called the neural crest. It has been proposed that melanocytes might be inherently predisposed to having invasive and metastatic attributes following neoplastic transformation as a result of their embryonic differentiation program. Metastatic melanoma cells are highly invasive and typically exhibit a high degree of plasticity, similar to their neural crest progenitors, but whether these cells recapitulate an embryonic invasion program related to their ancestral neural crest is unclear. We have shown that metastatic melanoma cells transplanted into the chick embryonic neural crest microenvironment invade into the host tissue, migrate along host neural crest cell migratory pathways, do not reform tumors, and can potentially be reprogrammed to a neural-crest-cell-like phenotype (Kulesa et al., 2006; Hendrix et al., 2007; Kasemeier-Kulesa et al., 2008). Here, we use the chick embryo neural crest microenvironment, combined with 2-photon photoactivation cell labeling and time-lapse microscopy, as a model to address the predisposition hypothesis in more detail. We find that melanoma cell orientation to the host cranial neural crest migratory routes and ellipticity of cell shape increase as cells stream out in distinct strand-like structures. Strand-like structures extend into the neural crest microenvironment, but give rise to individually invasive cells away from the neural tube. 2-photon photoactivation of PSCFP2, to discretely label small numbers of melanoma cells in ovo, reveals that the most invasive cells do not start at the tissue transplant periphery but may come from deep within the transplant. In vivo 2-photon time-lapse imaging confirms the unpredictable nature of melanoma dissemination and that some cells are more aggressively predisposed. These results indicate that melanoma metastasis resembles the neural crest migratory pattern but is unpredictable in the spatio-temporal order of invasion. We propose that the chick embryo transplant model is a valid, in vivo model of melanoma metastastasis.

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 5163.
Abstract 5169: The polycomb group protein BMI-1 cooperates with Yes-Associated Protein, YAP, to suppress cell contact inhibition in Ewing sarcoma cells
Authors: Hsu Jessie Hao-Ru , Hung Long , Lawlor Elizabeth R.
Published: February 2011

The polycomb group family protein BMI-1 is frequently deregulated in cancer. BMI-1 has been shown to promote stemness and tumorigenicity largely through epigenetic repression of the CDKN2A locus, inhibiting the expression of cell-cycle inhibitors p16INK4A and p14ARF. We have previously shown that BMI-1 functions as an oncoprotein in Ewing sarcoma, promoting anchorage independent growth in vitro and tumorigenicity in vivo independently of CDKN2A repression. In the current study we have investigated the potential contribution of BMI-1 to loss of cell contact inhibition, a fundamental transforming property of cancer cells. Using cells that stably express an shRNA against BMI-1, we discovered that loss of BMI-1 expression restores contact inhibition to CDKN2A-null Ewing sarcoma cells. Significantly, although proliferation of cells in log phase growth is unaffected by loss of BMI-1, at high cell density BMI-1 knockdown cells undergo cell cycle arrest and death. In contrast, control vector-transduced cells continue to enter cell cycle and proliferate, avoiding contact inhibition. Although many signaling pathways are involved in mediating the cell contact inhibition response, inactivation of the Yes-Associated Protein (YAP), a key downstream target of the Hippo pathway, has been implicated in both Drosophila and mammalian cells. Intriguingly, our data show that, although the Hippo pathway is activated in response to increasing cell density, as evidenced by increasing YAP phosphorylation, YAP is not degraded in control cells that express high levels of BMI-1. In contrast, silencing BMI-1 expression results in loss of YAP protein at high cell density coincident with induction of contact inhibition. Together, these findings suggest that YAP is a novel downstream target of BMI-1 and that BMI-1-mediated stabilization of YAP renders cancer cells impervious to cell contact inhibition. Experiments are currently ongoing to elucidate the precise molecular mechanism linking BMI-1 to YAP; however, preliminary studies show that knockdown of BMI-1 has no effect on YAP1 transcript expression at either low or high cell density indicating that the effects of BMI-1 on YAP stabilization are most likely to be post-translational and indirect. Cancer cells escape cell contact inhibition to achieve unrestrained proliferation and enhanced invasive and metastatic properties. Our findings suggest that BMI-1 may be a key upstream mediator of this critical cancer-associated 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 5169.
Abstract 5172: Role of CEACAM1 on cancer cell endothelial adhesion and liver metastasis in vivo
Authors: Chan Carlos H.F. , Arabzadeh Azadeh , DeMarte Luisa , Spicer Jonathan , Turbide Claire , Brodt Pnina , Beauchemin Nicole , Ferri Lorenzo E.
Published: February 2011

Background: There are emerging data to suggest that tumor over-expression of the carcinoembryonic antigen-related cell adhesion molecules (CEACAM1, 5 and 6) may influence cancer metastasis; however, the mechanisms for this are unclear. CEACAM1 was shown to bind homotypically to itself and heterotypically to CEACAM5 and CEACAM6. Since CEACAM1 is the only known CEACAM family member expressed on the hepatic endothelium, we hypothesized that CEACAM1 promotes cancer metastasis by directly mediating binding between circulating tumor cells and the sinusoidal endothelium.

Methods: Using intra-vital microscopy (IVM), a physiologically relevant model system to assess the early stages of liver metastasis in vivo, we have compared the hepatic endothelial adhesion of intra-splenically injected MC38 cells (mouse colon cancer cell line with minimal CEACAM1 expression) between Ceacam1+/+ and Ceacam1−/− mice. A CEACAM1-negative population of MC38 cells (MC38-null) was sorted out by FACS using specific anti-CEACAM1 antibodies. MC38-null cells were then infected with retroviruses produced from ψ2 packaging cells carrying pLXSN-CEACAM1 expression construct and infected cells were selected using neomycin-containing media. A CEACAM1-positive population of MC38 cells (MC38-CC1) was sorted out by FACS using specific anti-CEACAM1 antibodies. The migratory ability of MC38-null and MC38-CC1 cells were compared by IVM in Ceacam1+/+ mice as mentioned above. Liver metastasis assays were performed by means of intra-splenic injection of MC38 cells into the Ceacam1+/+ and Ceacam1−/− mice. Data expressed as mean ± SEM, student t-test determined significance (*p<0.05).

Results: By using IVM, we observed a 3-fold decrease in hepatic sinusoidal endothelial adhesion of intra-splenically injected MC38-null cells in Ceacam1−/− mice compared to Ceacam1 +/+ mice (0.8±0.2 vs 2.5±0.3 adhered cells)*. Forced expression of CEACAM1 in MC38 cells however did not significantly increase hepatic endothelial adhesion in the Ceacam1+/+ mice (2.7±0.9 adhered MC38-CC1 cells vs 2.5±0.2 adhered MC38-null cells). Development of liver metastasis after intra-splenic injection of MC38-null cells was significantly reduced in the Ceacam1−/− mice (81% reduction in comparison to Ceacam1+/+ mice)*.

Conclusion: Sinusoidal endothelial cell, but not cancer cell, CEACAM1 expression supports liver metastasis by increasing the adherence of circulating tumor cells to hepatic endothelium. Further investigation will be required to dissect the exact mechanism of how endothelial CEACAM1 influence hepatic endothelial adhesion of cancer cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5172.
Abstract 5177: Assessment of EpCAM function in lung cancer progression
Authors: Seung Han Jee , Kim Youngwook , Park Keunchil , Ahn Myung-ju
Published: February 2011

The epithelial cell adhesion molecule (EpCAM) is expressed on the basolateral membrane of the majority of epithelial tissues and it is upregulated or expressed de novo in various carcinomas. Several studies showed that high level EpCAM expression is associated with poor survival and tumor grading.

EpCAM remains an attractive cancer therapeutic target. Until recently, however, molecular impact of EpCAM on cancer signaling processes and growth regulation has not been clearly understood. We show here that inhibition of EpCAM by inducible shRNA resulted in a change of cell cycle profile and decreased cellular proliferation in non-small cell lung cancer cells. Cancer cells were apparently arrested in G1 phase, and subsequent western blotting experiments showed that p27 protein increased upon EpCAM inhibition.

To test whether EpCAM depletion would also lead to changes in tumor progression in vivo, we assessed the effect of EpCAM shRNA induction in lung cancer animal xenograft model. EpCAM knock-down led to almost complete tumor progression in vivo. Additional studies showed that inhibition of EpCAM expression was related to decreased microvessel density, revealed by CD31 immunohistochemical staining.

Overall, the data presented here suggest that EpCAM can be another therapeutic target of lung cancer. Development of inhibitory modality of EpCAM function needs to be tested further alone or in combination of other therapeutic reagents.

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 5177.
Abstract 5183: Correlation of cell adhesion molecules expression and prognosis in clinically localized prostate cancer
Authors: Júnior José Pontes , Ribeiro-Filho Leopoldo Alves , Reis Sabrina Thalita , de Oliveira Luiz Carlos Neves , Dall'Oglio Marcos , Leite Kátia R M , Srougi Miguel
Published: February 2011

Introduction and objective: E-cadherin and others adhesion molecules are responsible for the maintenance of the epithelial phenotype. Some studies have reported correlation between alterations in cell adhesion molecules expression and carcinogenesis, but their role in the prostate cancer is not clear. Our aim is to study the expression profiles of E-cadherin and catenins in localized prostate cancer and evaluate their association with recurrence after surgical treatment

Methods: For this case-control study, we selected 111 patients with localized tumor submitted to radical prostatectomy. 60 of these patients have not presented recurrence after a median follow-up of 123 months. Recurrence was defined as a PSA level above 0.4ng/ml. E-cadherin and α, β and µ-catenins expression was evaluated by immunohistochemistry in a Tissue Microarray containing two samples from each tumor. We employed a semiquantitative analysis and considered normal when the expression was strong and diffusely positive. We correlated expression with recurrence using the χ2 test and p<0.05 was considered significant.

Results: We observed normal expression for E-cadherin and α, β and µ-catenins in 77%, 42, 18% and 8% of patients respectively (figure 1). Correlation the expression of cell adhesion molecules with cancer recurrence after radical prostatectomy in the univariate analysis we found that these expressions were not associated with tumor outcome after surgery with more than ten years follow up (table1). Conclusions: In the present study we have shown a profile of cell adhesion molecules expression in localized prostate cancer who underwent surgery. Furthermore, we found that the expressions were not correlated with recurrence, which do not qualify these proteins as potential markers of prognosis for this tumor.

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 5183.
Abstract 5194: Induction of Fyn via the Ras/PI3K pathway is necessary and sufficient for enhanced migration and invasion
Authors: Yadav Vipin , Denning Mitchell F.
Published: February 2011

Over-expression and activation of Src family kinases, particularly Fyn, is relatively frequent in human cancers. Fyn promotes cancer progression, tumor survival and carcinoma invasion, making it a potential molecular target for cancer therapeutics; however the mechanism of Fyn over-expression in cancers is not clear. Since activation of Ras oncogenes in human cancers is a very common oncogenic event, we explored if an active Ras oncogene could induce Fyn expression. Interestingly, retroviral transduction of the immortalized human keratinocyte cell line HaCaT with oncogenic H-Ras dramatically upregulated Fyn mRNA, protein, and activity (>100 fold mRNA induction, p<0.001). Src levels and kinase activity were not affected by H-Ras transduction. Furthermore, activation of Akt, but not MAPK or EGFR, was necessary and sufficient for induction of Fyn by H-Ras. The enhanced migration and invasion induced by H-Ras could be significantly blocked (70% reduction, p<0.0001) by Fyn specific siRNA knockdown or inhibition by PP2 (10 µM). In addition, expression of active Fyn in HaCaT cells was sufficient for increased migration or invasion of cells. These results implicate induction of Fyn in Ras-induced enhanced migration and invasion of cancer cells. Focal adhesion kinase (FAK) activation has been strongly linked with the Ras-mediated increased migratory phenotype of tumor cells. We found that Fyn was necessary and sufficient for Ras induced activation of FAK. In summary, we show that Fyn induction by oncogenic Ras/PI3K/Akt pathway can account for the elevated Src family kinase activity in cancers, and is a critical mediator of the Ras-stimulated invasive cell phenotype. These results have strong implications for the development of therapeutic strategies targeting Akt/Fyn pathway to block migration and invasion of 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 5194.
Abstract 5198: Different functions of the chemokine receptor CXCR3 in normal and malignant mammary epithelial cells
Authors: Li Yanchun , Ma Xinrong , Kundu Namita , Goloubeva Olga , Holt Dawn , Fulton Amy M.
Published: February 2011

Our previous studies detected chemokine receptor CXCR3 in malignant epithelium of early stage breast cancers by immunohistochemistry and the expression levels were positively correlated with significantly poorer survival. We also showed that CXCR3 contributes to metastatic potential in a murine model of metastatic breast cancer. Interestingly, although immortalized normal murine and human mammary epithelial cells (EpH4 and MCF-10A) also express CXCR3, they respond to CXCR3 ligands differently than malignant cells. Migration of tumor cells was enhanced by stimulation with CXCR3 ligands, but a negligible chemotactic response to these ligands was observed in normal cells. Signal transduction activation patterns also differ between malignant and normal cells. In tumor cells, ERK1/2 is robustly activated after CXCR3 ligand stimulation, whereas the same pathway is inhibited in normal cells. Likewise the p38 pathway is activated in tumor cells and decreased in normal cells. Thus, CXCR3 activation alters the behavior of both malignant and normal cells, but in different ways. One possible mechanism to explain different functions of CXCR3 between normal and malignant mammary epithelial cells is the expression of different CXCR3 isoforms. It has been shown that the “classical” CXCR3 receptor (CXCR3-A) mediates proliferation and migration of various cell lines, while CXCR3-B, a splice variant of CXCR3-A, has opposing functions. To determine the expression pattern of these two CXCR3 isoforms in a panel of human breast cancer cell lines, we conducted real-time quantitative PCR and showed that the CXCR3-B/CXCR3-A mRNA expression ratio is decreased (0.16-0.63) compared with normal epithelial cell line MCF-10A (2.16). CXCR3-B expression dominates in MCF-10A cells, while CXCR3-A expression dominates in tumor cells, with the most metastatic cell line (MDA-MB-231) exhibiting the lowest CXCR3-B/CXCR3-A ratio (0.16). Taken together, these data indicate that the level of CXCR3 receptor expression is not the only determinant of function; the relative amounts of CXCR3-A and CXCR3-B may also be important. Further studies will elucidate how the balance between CXCR3-A and CXCR3-B contributes to the behavior of cells and allow us to optimize therapeutic strategies targeting CXCR3.

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 5198.
Abstract 5202: MUC1 oncogene is required for EGF/Src-driven carcinoma cell metastasis
Authors: Lau Steven K. M. , Shields David J. , Murphy Eric A. , Cheresh David A.
Published: February 2011

Epidermal growth factor receptor (EGFR) and Src tyrosine kinase signaling promote epithelial to mesenchymal transition (EMT) and have been associated with increased metastatic capacity of tumor cells. We observed that EGFR/Src signaling leads to the activation of integrin αvβ5 on carcinoma cells, resulting in an increased invasive capacity without influencing primary tumor growth in vivo (Ricono, et al 2009). We explored potential downstream effectors of Src that may influence this metastatic cascade using an in silico screening approach. Leads were selected based on their ability to influence EGFR/Src/αvβ5-dependent invasion in vitro and metastasis in vivo in a chicken chorioallantoic membrane model. Here, we report the transmembrane glycoprotein mucin-1 (MUC1) as an essential mediator of the EGFR/Src/αvβ5 signaling axis leading to the metastasis of carcinoma cells in vivo. Disruption of MUC1 expression selectively abolished EGF/Src/αvβ5-dependent migration in vitro and metastasis in vivo without influencing primary tumor growth. Importantly, EGFR- and Src-dependent phosphorylation of the cytoplasmic tail of MUC1 (MUC1-CT) facilitated cleavage of MUC1-CT. Once cleaved, MUC1-CT was able to associate with β-catenin, enter the nucleus and modulate gene expression relevant to the metastatic properties of these cells. These findings support a critical role for MUC1 in the regulation of EGFR/Src/αvβ5-dependent metastasis of a wide range of carcinomas.

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 5202.
Abstract 5204: Nodal regulates invasive phenotypes in extravillous and choriocarcinoma trophoblast cells
Authors: Zhang Guihua , Postovit Lynne Marie
Published: February 2011

Nodal, an embryonic morphogen and a member of the transforming growth factor-beta (TGF-ß) superfamily, is highly expressed in embryonic development and some cancers such as breast cancer and melanoma. It has been noticed that altered Nodal expression leads to changes in cellular invasiveness both during embryogenesis and cancer metastasis. However, the roles of Nodal in trophoblast invasion and tumor progression remain unclear. The present study characterizes and examines the effects of Nodal on cell migration, invasion and proliferation by using a highly invasive first trimester trophoblast cell line, HTR-8/svneo, and three Chrioncarcinoma cell lines as in vitro models.

By knocking down Nodal expression with small hairpin RNA (shRNA) plasmid DNA and morpholino oligomers, stable and transient Nodal knockdown clones were created. RT-PCR and Western Blots were performed and analyzed to verify Nodal knockdown in comparison to the Non-transfected (control) cells. To further confirm successful Nodal knockdown, downstream Nodal signalling (phospho-Smad2) was tested and analyzed by Western Blot. Nodal knockdown cells were then subjected to: (1) growth curves to evaluate the effect of nodal level on cell growth capacity and viability; (2) transwell migration assays to assess the effect of Nodal on cell movement and mobility; (3) transwell invasion assays to investigate the effect of nodal on cell invasive potential. The data show that knockdown of Nodal results in a decrease in cell migration and invasion, but does not significantly alter proliferation in vitro. Furthermore, the addition of human recombinant Nodal results in a restoration of cell migration and invasion. In order to further confirm the involvement of Nodal in trophoblast invasion, and to correlate Nodal levels to trophoblast cell phenotype, we then quantified the amount of Nodal in three chrioncarcinoma cell lines (Jar, Jeg-3 and Bewo) with Western Blot, and checked their migratory and invasive potential by Transwell migration and invasion assays. The data show strong positive correlation between Nodal protein level and the cell migration and invasion capacity. Our data indicate that Nodal does play a significant role in trophoblast cell migration and invasion, the two major processes of tumor progression. By studying the first trimester invasive trophoblast cell line, a greater understanding about the role of Nodal shared by trophoblast invasion and tumor metastasis may be gained.

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 5204.
Abstract 5206: VEGF-A/VEGFR-2 signaling might be a promising target for the tumor invasion of pancreatic cancer
Authors: Doi Yosuke , Yashiro Masakazu , Yamada Nobuya , Amano Ryosuke , Kato Yukihiro , Kashiwagi Shinichiro , Noda Satoru , Fuyuhiro Yuhiko , Komoto Masahiro , Matsuoka Junko , Kubo Naoshi , Muguruma Kazuya , Tanaka Hiroaki , Inoue Toru , Sawada Tetsuji , Ohira Masaichi , Nakata Bunzo , Hirakawa Kosei
Published: February 2011

Introduction: Pancreatic cancer is one of the most lethal solid tumors. Vascular endothelial growth factor receptors (VEGFRs) are expressed not only by endothelial cells but also by some types of cancer cells, including pancreatic cancer cells. VEGFRs might play an important role for the development of cancer cells. The purpose of this study was to evaluate the efficacy of VEGF/VEGFR-2 targeted therapy in pancreatic carcinoma. We here demonstrated that VEGFR-2 signaling on pancreatic cancer cells might be a promising therapeutic target for the invasion of pancreatic cancer.

Materials & Methods: Three pancreatic carcinoma cell lines, MiaPaCa-2, OCUP-AT and Panc-1, were used. The effect of anti-VEGF antibody, bevacitumab, a tyrosine kinase inhibitor against VEGFRs, sunitinib, and VEGF-R2 siRNA on cancer invasion was examined by invasion assay. The expression level of VEGFR mRNA of cancer cells was examined by RT-PCR. The effect of VEGF, bevacitumab and sunitinib on MAPK and PI3K signaling pathway was examined.

Result: VEGF-A significantly increased the number of migration cells of MiaPaCa-2 and OCUP-AT, but not that of Panc-1. Bevacitumab and sunitinib significantly decreased the migration ability of MiaPaCa-2 and OCUP-AT cells. The expression levels of VEGF-R2 were high in MiaPaCa-2 and OCUP-AT cells, in compared with Panc-1 cells. Invasion and migration ability of MiaPaCa-2 cells significantly (p<0.05) decreased by VEGFR-2 siRNA treatment. The level of VEGFR-2 autophosphorylation, p-ERK and p-Akt expression was increased by VEGF-A, and decreased by bevacitumab and sunitinib. Conclusion: The invasion of pancreatic cancer cells was closely associated with VEGF-A/VEGFR-2 signaling. VEGF-A/VEGFR-2 inhibition by bevacitumab or sunitinib appears therapeutically promising in pancreatic cancer with VEGFR-2 expression.

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 5206.
Abstract 5209: Abrogation of STAT3 activation by JSI-124 enhances dasatinib-induced cytotoxicity in malignant human glioma cell lines
Authors: Jane Esther P. , Premkumar Daniel R. , Agostino Naomi R. , Scialabba Joseph L. , Pollack Ian F.
Published: February 2011

Src family kinases (SFK) collectively regulate a variety of cellular functions in many cancer types including proliferation, invasion, motility, survival, differentiation, and angiogenesis. Src greatly enhances EGFR-mediated transformation, raising the possibility that Src and/or its related family members may be effectors of EGFR signaling in cancer, including in glioblastoma. Although Dasatinib (BMS-354825), an ATP-competitive, small molecule tyrosine kinase inhibitor, suppresses the activity of SFKs at nanomolar concentrations, IC50 values ranged from 7 to 30 µM for inhibition of proliferation, well above the clinically achievable range. Because Src kinases were initially implicated in STAT activation, we hypothesized that the combination of Src inhibitor dasatinib and JAK2/STAT3 inhibitor JSI-124 would promote glioma cytotoxicity by decreasing both the activation status of c-Src and STAT3, as well as downregulating the levels of other relevant signaling effectors. We, therefore, examined the effects of dasatinib and JSI-124, alone or in combination, on signal transduction, migration and apoptosis in a series of malignant human glioma cell lines. JSI-124 significantly enhanced the efficacy of dasatinib in vitro. Combination of dasatinib and JSI-124 displayed a significant inhibition of cell migration in all cell lines. The effects on cell migration correlated with the inhibition of Src and downstream mediators of adhesion (e.g. focal adhesion kinase). Cells exposed to dasatinib and JSI-124 induced morphological changes that were consistent with an upstream role for Src in regulating focal adhesion complexes. Taken together, targeting Src and STAT pathway may contribute to the treatment of cancers that demonstrate increased levels of EGFR, Src and STATs, including malignant human glioma

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 5209.
Abstract 5214: Role of mitogen-activated protein kinase (ERK) signaling in cyclooxygenase 2-mediated colon cancer cell migration
Authors: Hessel Kara , Bean Ronald R. , Jakobi Rolf , Chang Yu-Wen E.
Published: February 2011

Colon cancer is the second most common cause of cancer-related deaths. Many patients present with colon cancer in advanced stages of metastasis, where the outcome is poor and mortality is high. Invasion is an essential capability acquired by cancer cells to metastasize to distant organs. Currently there are no effective strategies to prevent invasion because the underlying mechanisms are poorly understood. During early development of colon cancer, cyclooxygenase 2 (COX-2) is constitutively induced. We have shown that COX-2 promotes invasion by disrupting cell adhesion and increasing motility in HCA-7 colon cancer cells that constitutively express endogenous COX-2. To further determine the mechanisms by which COX-2 stimulates motility and to confirm this cellular behavior in other colon epithelial cells, we established HCT-15 colon cancer cells that stably express recombinant COX-2 (HCT-15-COX-2). Scratch wound gap assays were used to monitor cell motility and proliferation in HCA-7 and HCT-15-COX-2 cells. HT-29 colon cancer cells that do not express COX-2 and parental HCT-15 cells were used as controls. HCA-7 and HCT-15-COX-2 cells closed the gap significantly faster than HT-29 and HCT-15 cells. Inhibition of COX-2 in HCA-7 and HCT-15-COX-2 cells by the COX-2 selective inhibitor NS-398 decreased the gap closing. Interestingly, the enhanced gap closing ability of HCA-7 and HCT-15-COX-2 cells correlated with activation of ERKs, mitogen-activated protein kinases that are known to mediate COX-2 signaling to stimulate colon cancer cell growth. Activation of ERK depended on COX-2 activity. Inhibition of ERK signaling in HCA-7 and HCT-15-COX-2 cells reduced gap closing as effectively as NS-398. Furthermore, inhibition of ERK signaling prevented migration of both HCA-7 and HCT-15-COX-2 cells in modified Boyden chamber assays. Interestingly, reduced migration by inhibition of ERK signaling correlated with increased protein levels of adherens junction protein alpha-catenin. Our data reveal for the first time a role for ERK to promote cell migration in response to COX-2 signaling in addition to its stimulation of cancer cell growth. Since cardiovascular toxicity caused by long-term use of COX-2 inhibitors raises several concerns, targeting ERK can be an alternative approach for preventing colon cancer growth and invasion.

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 5214.
Abstract 522: Comparison of ovarian cancer spheroids and three-dimensional cell movement with the spheroids formed by normal ovarian surface epithelial and fallopian tube epithelial cells using an in vitro culture system
Authors: Choi Pui-Wah , Megan Hanna , Fong Wing-Ping , Ng Shu-Wing
Published: February 2011

Ovarian cancer is the most lethal cause of death among gynecological cancers. Recent studies have suggested that the cells of origin of ovarian cancer might be the ovarian surface epithelial (OSE) cells and epithelial cells derived from other Mullerian ducts such as distal fallopian tube. It is believed that these cells exist as cortical inclusion cysts in the ovary, to which the extracellular matrix (ECM) proteins, proteases, secreted growth factors and chemokines from the stromal microenvironment can provide oncogenic signals in a paracrine fashion. In order to investigate the molecular mechanisms and signaling events governing these processes, we grew ovarian cancer cell lines, normal OSE and fallopian tube epithelial (FTE) primary cells as three-dimensional (3D) cultures in Matrigel, which was enriched with basement membrane proteins. Most of the cells formed spheroid structures resembling ovarian inclusion cysts, with sizes ranging from 50 to 100 μm. One cancer cell line formed spindle-like structure. The multicellular structures were stained for membrane markers and examined using confocal laser scanning microscopy. Most of the cancer spheroids were consisted of tightly packed polarized epithelial cells, as revealed by lateral β-catenin and E-cadherin staining, and GM130 staining at the apical surface outlining a central lumen. In contrast, normal OSE spheroids and FTE spheroids showed less organized structure with random β-catenin and GM130 staining. To evaluate the growth and progression of the cancer cells in response to stimuli from a stromal microenvironment, the spheroids were transferred to a collagen matrix. Interestingly, the majority of the cancer cells migrated out of the spheroids in a collective movement similar to the lymphatic metastasis observed in breast cancer. The morphology of the collectively moving cells was distinctively different from that expected from an epithelial-mesenchymal transition model. Ongoing gene expression profiling and characterization of the cells growing in the 3D spheroids will reveal the molecular mechanisms underlying the morphogenic changes and may provide insights into the growth and progression of clinical ovarian cancer from inclusion cysts.

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 522.
Abstract 5221: Angiogenesis Imaging of Orthotopic Bladder Tumors using a Smart MRI Contrast Agent Containing VEGF121/rGel
Authors: Yang Jaemoon , Cho Eun-Jin , Mohamedali Khalid A. , Farhangfar Carol J. , Hittelman Walter N. , Suh Jin-Suck , Rosenblum Michael G. , Huh Yong-Min
Published: February 2011

Comprehension of the angiogenic vessels from solid tumors and metastatic lesions is a crucial step for the treatment of cancer. In particular, recognition of VEGFR-2 (KDR) as primary proangiogenic factors and their distribution analysis is very important for establishment of the treatment strategy. In this research, we thus formulated the smart imaging probes based on VEGF121/rGel conjugated MnFe2O4 for imaging of tumor neovasculature using an orthotopic bladder tumor xenograft model. VEGF121/rGel was conjugated to MnFe2O4 nanoparticles (MNPs). Their colloidal size was around 45 nm and a high relaxivity coefficient was 423 mM−1s−1. As in vitro test, the targeting efficiency and detection capability of the VEGF121/rGel-MNPs were investigated in both KDR deficient (253JB-V) and KDR-overexpressing (PAE/KDR) cell lines using MRI, respectively. The targeting and internalization of VEGF121/rGel-MNPs into cells was confirmed by electron microscopy. Their phosphorylation ability and cytotoxicity were compared to unconjugated VEGF121/rGel. They demonstrated astonishing targeting capability against KDR-overexpressing PAE/KDR cells, as confirmed by dose-dependent MR images and VEGF121 inhibition tests. The phosphorylation activity of KDR and cytotoxicity of VEGF121/rGel-MNPs were evaluated. The orthotopic tumor mouse model was established by implanting low KDR-expressing 253JB-V cells into the bladder dome. After tail-vein injection of VEGF121/rGel-MNPs, the MR signal enhancement of intratumoral vessels by VEGF121/rGel-MNPs was observed and inhibition test using VEGF121 was also conducted. VEGF121/rGel-MNPs successfully targeted the tumor and provided accurate anatomical details through: i) acquisition of clear neoangiogenic vascular distributions and ii) obvious enhancement of the MR signal in T2*-weighted images. Immunostaining and blocking studies demonstrated the specific targeting ability of VEGF121/rGel-MNPs towards intratumoral angiogenesis. Furthermore, ex vivo MR imaging of tumor tissue, and a CD-31 and rGel fluorescence immunostaining study revealed the targeting ability to VEGFR-2 on the angiogenetic vessels. Consequently, synthesized VEGF121/rGel-MNPs as valuable MR imaging contrast agents can be specifically delivered to tumors and bind to KDR-expressing angiogenic tumor vessels.

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 5221.
Abstract 5222: Molecular imaging of GSK-3β and CK1α kinase activities in response to Wnt signaling
Authors: Nyati Shyam , Bhojani Mahaveer Swaroop , Pasupulathi SwathiChitra , Ross Brian D. , Rehemtulla Alnawaz
Published: February 2011

The Wnt signaling pathway has been found to be disregulated in many carcinomas of gastrointestinal system. GSK-3β and CK1α act as molecular switch that dictates both on and off states of Wnt/beta-catenin signaling by regulating cellular levels of the Wnt effector molecule β-catenin. In the absence of the Wnt signal β-catenin is initially phosphorylated by CK1α at S45, followed by GSK-3β at S41, T37 and S33 which marks it for proteosomal degradation. Although GSK-3β is one of the key regulators of the Wnt signaling pathway, the ability for direct, non-invasive & real-time measurement of its kinase activity has been unavailable. Here we describe a chimeric, gain of function, split luciferase based novel reporter for monitoring of dual kinase (GSK-3β and CK1α) activity.

The N-terminal β-catenin peptide (aa 29-48) harboring phosphorylation sites for GSK-3β and CK1α was synthesized and used to construct bioluminescence β-catenin reporter (BCR). This reporter when expressed in SW620 colon carcinoma cells and treated with LiCl and small molecular inhibitors SB415286 (GSK-3β) and CKI-7 (CK1α), lead to a dose and time dependent increase in the bioluminescence which was validated by western blotting. Cell lines stably expressing mutant reporters exhibited attenuated response to LiCl & SB415286 (S37A mutant) and to CKI-7 (S45A mutant) confirming specificity of the reporter. To quantify the Wnt dependent change in the activity of the reporters, stable cell lines were generated in HEK293 background which has an intact Wnt signaling pathway. The WT reporter cell line showed over two fold induction with Wnt conditioned media while S37A mutant reporter cell line did not show a significant change in reporter activity corroborating the specificity of the reporters.

In mice bearing tumors expressing the wild-type reporter, treatment with LiCl resulted an initial increase in the bioluminescence activity at 4 h which peaked at 7 h. Subsequent imaging revealed oscillations of the bioluminescence activity with a frequency of 14 h and an amplitude that decreased with time. Our reporters provide a powerful, novel tool for sensitive, real time, dynamic, non-invasive monitoring of GSK-3β and CK1α kinase activities in-vitro and in-vivo and have the potential in executing novel therapeutic regimens targeting GSK-3β/CK1α.

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 5222.
Abstract 5225: Pemetrexed induced thymidylate synthase inhibition in non-small cell lung cancer in vivo: a pilot study with 18F-FLT PET
Authors: Frings Virginie , van der Veldt Astrid AM , Lubberink Mark , Herder Judith , Peters Godefridus J. , Honeywell Richard , Thunnissen Frederik BJM , Hoekstra Otto S. , Smit Egbert F.
Published: February 2011

Objectives Thymidylate synthase (TS) is a key enzyme for de novo synthesis of DNA and as such a target for anticancer drugs. In a mouse model, effective TS inhibition resulted in a 1.8 fold increase of 3′-deoxy-3′-[(18)F]fluorothymidine (18F-FLT) cellular uptake (Cancer Res 2006;66:8558-64). Here we investigated changes in 18F-FLT uptake 4 hours after the first dose of pemetrexed in patients with non-small cell lung cancer (NSCLC).

Methods Twelve NSCLC patients (mean age 64 yrs, 4 male) treated with pemetrexed (in combination with cisplatin, carboplatin or monotherapy) were included. Patients underwent dynamic 18F-FLT PET at baseline and 4 hours after the first therapeutic dose of pemetrexed. For primary tumors, regions of interest (ROI) were defined with a 41, 50 and 70% threshold of the maximum pixel, and the maximum standardized uptake value (SUVmax) was determined (as well as full kinetic analysis). One, 2, 3, 4 and 6 hours after pemetrexed administration, blood samples were collected to measure deoxyuridine levels using LCMSMS. After six weeks, RECIST was used to evaluate tumor response. Association between 18F-FLT uptake and RECIST was tested with a chi-square test.

Results According to RECIST 3 patients had PR, 7 SD and 2 PD. Four hours after administration of pemetrexed, two patients had increased 18F-FLT uptake (beyond test-retest borders, 22% for SUVmax), while three patients had a significantly decreased uptake (see Table). In 7 out of 12 patients, the change in 18F-FLT uptake was within the test-retest variability. In all patients deoxyuridine levels raised after administration of pemetrexed with a average maximum at 4 hours. There was no significant association between 18F-FLT uptake and RECIST.

Conclusions Measuring TS inhibition in a clinical setting 4 hours after pemetrexed revealed a non-systematic change in 18F-FLT uptake. The association with response measurement on CT was not significant.18F-FLT PET and RECIST per patientPt No.Baseline 18F-FLT uptake (SUVmax)18F-FLT uptake 4 hours after pemetrexed (SUVmax)Change 18F-FLT in uptake (%)RECIST after 6 weeks14.74.94.1SD24.44.75.0SD34.12.3−44.7SD44.04.411.0PD53.54.528.6PR63.42.2−33.3SD74.85.05.7SD812.57.9−36.6PR911.312.17.1PD105.67.228.3SD115.15.35.7PR124.73.7−21.5SD

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 5225.
Abstract 5230: In vivo imaging of tumor malignancy with near-infrared fluorescence probes specific to HIF-active cells
Authors: Kuchimaru Takahiro , Kadonosono Tetsuya , Ushiki Takashi , McDougall Mark , Kizaka-Kondoh Shinae , Hiraoka Masahiro
Published: February 2011

In solid tumors, the distribution of oxygen pressure is not homogenous because of the uncontrolled tumor growth and immature blood vessels during angiogenesis, which generate a hypoxic microenvironment. Tumor hypoxia causes resistance to radiotherapy and chemotherapy, and malignant progression Hypoxia-inducible factor-1 (HIF-1) is a master transcriptional regulator for adaptation to hypoxia by inducing more than 100 genes, which are closely associated with malignant phenotype. Thus, HIF-1-active cells are hallmark of malignant tumors. We have been developing fusion protein probes specific to HIF-1-active microenvironment for imaging and targeting malignant tumors. We recently constructed a fusion protein POH, which consisted of Protein Transduction Domain (PTD), Oxygen-dependent Degradation Domain (ODD) and HaloTag. The PTD was a membrane-permeable peptide, which efficiently delivered fusion proteins into cells. The ODD was responsible for the oxygen-dependent regulation of the probe, which stabilized in a hypoxic environment and degraded immediately under normoxic conditions. In addition, HaloTag was used to covalently conjugate with its specific ligand labeled with near-infrared fluorescence (NIRF) dye. POH-NIRF was examined for its target-specificity and in vivo dynamic status by in vivo and ex vivo fluorescence imaging with IVIS-SPECTRUM. To visualize the targets in vivo, we used bioluminescence imaging of tumor cells, which stably retained a HIF-1-dependent luciferase reporter gene. POH-NIRF probes successfully imaged HIF-1-active regions defined by bioluminescence imaging in subcutaneously implanted human xenograft tumors and orthotopic pancreatic cancers 9 to 24 hrs after POH-NIRF injection. Furthermore, immunohistochemical analysis of tumor sections from the mice 6h after POH-NIRF injection revealed the regions imaged with POH-NIFR was overlapped with HIF-1α-positive regions, while the intracellular localization of POH-NIRF was in cytoplasm. Overall results demonstrated that POH-NIRF was a specific probe for HIF-1-active/hypoxic cells and suggested that a PTD-ODD probe would become a unique imaging probe for tumor malignancy.

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 5230.