bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2370: A novel Src/Ezrin pathway regulates local invasion and metastasis of breast
Authors: Mak Hannah , Naba Alexandra , Varma Sonal , Meens Jalna , Arpin Monique , SenGupta Sandip , Elliott Bruce E.
Published: February 2011

The membrane cytoskeletal crosslinker, ezrin, is frequently over-expressed in human breast cancer, and is required for motility and invasion of epithelial cells. Our group recently showed that ezrin acts co-operatively with the non-receptor tyrosine kinase, Src, in transformation of epithelial cells, requiring phosphorylation of specific tyrosines on ezrin by Src (Srivistava et al. MBC, 16:1481-90, 2005; Naba et al. EMBO J. 27:38-50, 2008). We showed that Src binds to ezrin Y190 via its SH2 domain and that ezrin Y145 and Y477 are phosphorylated by Src. Expression of Y145F or Y477F ezrin mutants, which cannot be phosphorylated by Src, delays cell spreading of epithelial cells on fibronectin substratum. Furthermore, ezrin is required for metastasis in a breast tumor xenograft model (Elliott et al. BCR 7:R365-73, 2005). We therefore examined whether Src/ezrin interaction regulates invasion and metastasis of breast cancer, using a mouse breast tumor (AC2M2) xenograft model. Transplanted tumor cells expressing vector alone rapidly infiltrated into surrounding stroma, underlying abdominal muscle, visceral organs and metastasized to lung and other sites. Interestingly, both Y145F and Y477F ezrin mutants had little effect on primary tumor growth rate. However, Y145F ezrin showed a marked attenuating effect on pulmonary metastasis. Furthermore, transplanted mouse breast carcinoma cells over-expressing Y477F ezrin formed cohesive tumors that were circumscribed by normal stroma with no detectable infiltration into underlying muscle. Marked lymphovascular involvement, a prognostic indicator of relapse in clinically advanced human breast cancers, was evident in control tumors but not in tumors expressing Y477F ezrin. In a preliminary IHC analysis, total ezrin showed strong cytoplasmic and weak membranous staining in the cortical region of control primary tumors, but showed strong membranous and weak cytoplasmic staining in Y477F ezrin-expressing primary tumors. The cohesive phenotype of Y477F ezrin is likely a consequence of a defect in cell adhesion as described by Naba et al. Thus, the precise state of ezrin phosphorylation may elicit different effects on local invasion and metastasis. Our study implicates a role of the Src/ezrin pathway in regulating local invasion and metastasis of breast carcinoma cells, and provides a clinically relevant model for assessing the Src/ezrin pathway as a potential prognostic marker and treatment target for invasive breast cancer. (Funded by CBCRA 17374 and ARC 4823)

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 2370.
Abstract 2372: The metastasis efficiency modifier ribosomal RNA processing 1 homologue (RRP1B) is a chromatin-associated factor
Authors: Crawford Nigel P.S. , Yang Hailiu , Mattaini Katherine R. , Hunter Kent W.
Published: February 2011

Previous experiments using the polyoma middle T (PyMT) mouse model of mammary tumorigenesis have demonstrated that host germline variation modulates metastasis in breast cancer. Recent work using the PyMT model has identified novel metastasis susceptibility genes, an example of which includes Rrp1b (ribosomal RNA processing 1 homolog B). Over-expression of Rrp1b in a mouse mammary tumor cell line induces a gene expression signature that predicts survival in a well characterized Dutch breast cancer dataset. Additionally, a polymorphism was identified within human RRP1B (G1421A; P436L), which is associated with differential survival in multiple breast cancer cohorts.

Here we extend the analysis of RRP1B function by demonstrating that the Rrp1b activation gene expression signature accurately predicts survival in three additional publicly available breast carcinoma gene expression datasets. We have also investigated the mechanism of action of RRP1B by defining protein binding partners for this germline-encoded metastasis modifier. Tandem affinity purification demonstrated that RRP1B physically interacts with many nucleosome binding factors, all of which have been implicated in regulation of gene expression. Examples include the linker histone H1X, poly(ADP-ribose) polymerase 1, tripartite motif-containing 28 (TIF-1β/KAP1), and cold shock domain protein A. These interactions were confirmed by co-immunofluorescence (Co-IF) and co-immunoprecipitation (Co-IP). Additionally, co-IF experiments demonstrated that RRP1B is localized to the perinuclear and perinucleolar regions, which is a particularly interesting given that factors localized to these regions are often involved in heterochromatinization and gene silencing. Furthermore, co-IP and co-IF demonstrated that RRP1B interacts with heterochromatin protein-1α and acetyl-histone H4 lysine 5, suggesting that RRP1B may well regulate gene expression through interactions with heterochromatin and euchromatin.

Finally, we investigated the effects of ectopic expression of the 436Pro and 436Leu RRP1B allelic variants that have previously been associated with differential survival in breast cancer. Gene expression analyses demonstrate that, compared with ectopic expression of wildtype 436 Pro RRP1B in HeLa cells, the variant 436Leu RRP1B differentially modulates various transcription factors controlled by TRIM28 and CSDA. These data suggest that RRP1B, a tumor progression and metastasis susceptibility candidate gene, is potentially a dynamic modulator of transcription and chromatin structure.

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 2372.
Abstract 2376: Endothelial cells acquire abnormalities by factors from tumor cells
Authors: Kosuke Akiyama , Ohga Noritaka , Hida Yasuhiro , Akino Tomoshige , Hirata Naoya , Takuro Kurosu , Ishikawa Shuhei , Kondo Miyako , Maishi Nako , Kawamoto Taisuke , Inoue Nobuo , Shindoh Masanobu , Hida Kyoko
Published: February 2011

Recently, it has been reported that tumor microenvironment is important for tumor progression and metastasis. We have reported that tumor endothelial cells (TECs) differ from their normal counterparts, in many aspects. For example, TECs upregulate several genes, have cytogenetical abnormalities and respond differently to the growth factors such as epidermal growth factor receptor (EGFR) or some anticancer drugs (Hida et al., Cancer Res 2004, Cancer Sci 2008). In this study, we focused on the interaction between tumor cells and endothelial cells to address the mechanism of TEC abnormality.

We demonstrate that cultured conditioned medium (CM) from melanoma cells cause several phenotypic changes in Human Microvascular Endothelial Cells (HMVEC). The cell proliferation of HMVEC was stimulated by melanoma CM. Furthermore, the expression level of Multi Drug Resistance Gene (MDR) was upregulated by melanoma CM treatment and HMVEC got less sensitive to Paclitaxel. It was suggested that some factors released from melanoma cause abmormalities in HMVEC phenotype. The study to elucidate mechanisms of these results are ongoing.

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 2376.
Abstract 2377: Aminopeptidase A plays a role in VEGF-induced migration and proliferation through direct activation by VEGFR2 kinase activity
Authors: Sikkema Arend H. , de Bont Eveline S. , Diks Sander H. , Elst Arja ter , Ruijtenbeek Rob , Boender Piet J. , Hoving Eelco W. , Kamps Willem A. , den Dunnen Wilfred F. , Peppelenbosch Maikel P.
Published: February 2011

Tyrosine kinases pose attractive targets for cancer therapy, as their abnormal signaling activity results in enhanced proliferation, migration and angiogenesis. Previously we have been able to generate a comprehensive description of the biological kinase activity present in pediatric brain tumor tissue lysate by the application of a peptide microarray containing 144 different tyrosine kinase peptide substrates. To establish which peptide substrates present on the array are most sensitive and discriminative for VEGFR activity we measured the peptide phosphorylation applying recombinant VEGFR1, 2 and 3. Furthermore, a panel of recombinant kinases with highly diverse VEGFR sequence homology was included to study substrate specificity.

An aminopeptidase A (APA) derived peptide proved to be highly phosphorylated by VEGFR 1 and 2. No phosphorylation of this peptide could be observed applying any of the other kinases, arguing that this peptide is discriminative for VEGFR1 and 2 activity. In the peptide array data of pediatric brain tumors, phosphorylation of the APA peptide was observed, suggestive for VEGFR activation.

Based on these results we hypothesize that APA can be activated through direct phosphorylation by VEGFR2 and exert functional effects on endothelial cell proliferation and migration. To test this hypothesis we stimulated human umbilical vein endothelial cells (HUVECs) with VEGF and observed phosphorylation of Aminopeptidase A.

Literature states that inhibition of APA suppresses VEGF-induced migration and proliferation of endothelial cells and inhibits angiogenesis. Based on our data we hypothesize that these APA-mediated effects of VEGF stimulation are induced through direct activation by VEGFR2 kinase activity.

Concluding, peptide microarray technology is able to provide a selective view on tyrosine kinase activation, as has been shown here for VEGFR1 and 2.

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 2377.
Abstract 2379: Rap1 regulates angiogenesis in prostate tumors
Authors: Menon Jyotsana , Doebele Robert S. , Schulze-Hoepfner Frank T. , Rosner Marsha Rich
Published: February 2011

Prostate cancer is the most frequently diagnosed malignancy in men and the second leading cause of cancer related deaths in the United States of America. Advanced stage prostate cancer is associated with poor prognosis since no effective therapies are available when the disease has progressed into a hormone refractory state. This grim prognosis indicates the need for novel approaches to the treatment of advanced stage prostate cancer. One such therapeutic strategy is anti-angiogenic therapy, which interferes with the blood supply of the tumor instead of targeting tumor cells directly. Here we focus on the role of Rap1, a small GTPase homologous to Ras, and its upstream activator, Epac, a guanine nucleotide exchange factor for Rap1, in angiogenesis. In previous studies, we showed that treatment of primary human microvascular endothelial cells (HMVEC) with 8 CPT-2Me-cAMP (8-CPT), a specific agonist of Epac, or introduction of constitutively active Rap1 into these cells blocked VEGF-mediated chemotaxis and inhibited angiogenesis in mice. To test the efficacy of Epac/Rap1 activation in a tumor model, we injected an Epac agonist, 8-CPT, in a PC-3 human prostate tumor xenograft model. Surprisingly, although 8-CPT did not alter tumor growth in PC-3 cells, the drug significantly reduced tumor size in PC-3 cells expressing constitutively active Rap1. Tumor analysis showed an increase in HIF-1α,VEGF and CD31 immunostaining in Rap1-expressing tumors, and a significant decrease in their levels upon treatment of the mice with the Epac agonist 8-CPT. Similar reductions in VEGF and Glut-1 mRNA levels and HIF-1α protein were observed in Rap1-PC-3 cells following 8-CPT treatment when cells were cultured under hypoxic conditions. On the basis of these results, we propose that expression of Rap1 in the PC-3 human prostate tumor cell line promotes hypoxic induction of angiogenesis and increases the susceptibility of the cells to anti-angiogenic agents.

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 2379.
Abstract 2390: Serotonin signal transduction in human endothelial cells
Authors: Zamani Ali , Roy Anshu , Zhai Ling , Li Rangbao , Qu Zhican
Published: February 2011

Angiogenesis is a highly regulated biological complex process, which consist of endothelial cells proliferation, migration, and tubulogenesis as key cellular steps to form new blood capillaries from preexisting vessels. Imbalance in angiogenesis contributes to a variety of human diseases. Tumor growth depends on angiogenesis and antiangiogenic agents have been developed as effective anti-cancer medicine. Recently, we have demonstrated that serotonin (5-HT), a well-recognized neurotransmitter, has a promoting effect on endothelial cells proliferation, migration and tubulogenesis, suggesting serotonin plays a role in regulation of angiogenesis. To study the role of serotonin in angiogenesis, we examined the expression of serotonin receptor isoforms in human endothelial cells. Real time PCR studies have showed that, serotonin receptor 1B (HTR1B), 1D (HTR1D), 1A (HTR1A) and 2B (HTR2B) are expressed in primary human endothelial cell lines. Blocking serotonin 1B receptor had significant inhibitory effect on endothelial cells proliferation and tubulogenesis. We have further explored the 1B-mediated signal transduction in endothelial cells. The results have revealed that the serotonin 1B receptor as a G protein-coupled receptor (GPCR) is coupled to Gi protein and decreased intracellular cAMP level upon its receptor binding. Both serotonin and 1B receptor agonist were able to activate ERK, p38, Akt, RhoA, Rac1 and PYK2/FAK signaling in endothelial cells. In contrast, a selective antagonist for serotonin 1B receptor blocks the serotonin-stimulated Akt and PYK2/FAK activation. Experiments with pertussis toxin (general Gi blocker) further demonstrated that heterotrimeric Gi-protein is required for the activation of Akt and PYK2/FAK by serotonin. These findings suggest that Akt and PYK2/FAK are involved in signaling transduction in serotonin-activated endothelial 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 2390.
Abstract 2401: IGF-IR peptide vaccine inhibits tumor growth via IFN-g-dependent biologic remodeling of the tumor
Authors: Cecil Denise L. , Park Kyong Hwa , Gad Ekram , Disis Mary L.
Published: February 2011

The insulin-like growth factor (IGF) pathway plays an important role in breast cancer growth and metastasis. The majority of breast tumors overexpress the IGF-I receptor (IGF-IR). Increased IGF-IR signaling in breast cancer is correlated with a poor prognosis and is associated with disease that is resistant to chemotherapy, radiotherapy, and biologic therapy. Thus, therapeutically targeting the IGF-IR signaling pathway is a promising approach to treat breast cancer. We have determined that the IGF-IR is immunogenic in breast cancer. Furthermore, vaccination with MHCII IGF-IR-specific peptides p384-398, p575-589, p951-965, and p1122-1136 induces a CD4+ T cell-dependent anti-tumor immune response in Neu-transgenic mice. Preliminary data suggests that IGF-IR specific immunization results in both decreased proliferation and increased apoptosis of tumor cells. Antigen specific CD4+ T cells secrete IFN-g and we have demonstrated that the inhibition of tumor growth with the IGF-IR vaccine is dependent on IFN-g. We questioned the mechanism whereby IFN-g-secreting CD4+ T cells targeting IGF-IR could inhibit tumor growth. Studies suggest that tumor-associated IGF-IR activation is decreased in tumors from vaccinated mice, with concomitant upregulation in SOCS-3, which has been shown to be modulated by IFN-g. In addition, IFN-g stimulation of the tumor cell line derived from the Neu-transgenic mouse decreased IGF-IR activation and induced SOCS-3 expression. Thus, active immunization targeting IGF-IR may induce both immunologic and biologic effects resulting in inhibition of breast cancer growth.

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 2401.
Abstract 2404: A DNA vaccine encoding the androgen receptor ligand binding domain elicits anti-tumor immune responses in vivo
Authors: Olson Brian M. , Johnson Laura E. , McNeel Douglas G.
Published: February 2011

The androgen receptor (AR) is a nuclear receptor that plays a critical role in both androgen-dependent and -independent prostate cancer, and remains the primary target of therapy for metastatic prostate cancer. We have shown that the AR may be a potential immunological target antigen, identifying some prostate cancer patients that have concurrent antibody and cellular immune responses specific for the AR ligand-binding domain (LBD). We have also identified that some patients have cytotoxic T lymphocytes (CTL) that are specific for ARLBD-derived HLA-A2 epitopes, some of which can lyse prostate tumor cell lines. Moreover, we demonstrated that these epitope-specific CTL could be elicited in HLA-A2 transgenic mice by means of a DNA vaccine encoding the ARLBD (pTVG-AR). In this report, we sought to evaluate whether this DNA vaccine could elicit anti-prostate tumor responses in relevant rodent models in which the ARLBD is expressed as a “self” antigen. First, Copenhagen rats immunized with the pTVG-AR vaccine four to six times at 14-day intervals were challenged with a syngeneic prostate tumor cell line subcutaneously implanted. These animals were found to develop T-cell responses specific for the ARLBD and have a significant delay in tumor growth compared with control vaccinated animals (p=0.018). In a separate autochthonous model, HLA-A2 transgenic mice were crossed with TRAMP mice and the resulting transgenic hybrid (A2/TRAMP) expressed HLA-A2 and developed prostate tumors driven by the SV40 large T antigen under a prostate-specific promoter. A2/TRAMP mice were similarly immunized with pTVG-AR, or control vector, at 14-day intervals and evaluated at 16 weeks of age. Mice immunized with pTVG-AR were found to develop HLA-A2 epitope-specific CD8+ T cell responses, and had fewer well-differentiated adenocarcinomas compared with control immunized mice. Finally, A2/TRAMP mice were immunized repetitively over time and followed for survival. Mice immunized with pTVG-AR (n=8) had a median survival of 218 days, compared with a median survival of 198 days for control (n=7) immunized animals (p=0.040, log-rank test). These combined data show that not only can the ARLBD be safely targeted in vivo (none of the immunized animals had any sign of autoimmunity or overt toxicity), but more significantly that vaccines targeting the ARLBD can elicit an anti-prostate tumor responses in separate animal models. These findings suggest that the ARLBD is a potentially attractive immunological target antigen for the treatment of human prostate cancer.

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 2404.
Abstract 2408: Selective delivery of NY-ESO-1 antigen protein by cholesteryl pullulan (CHP) to CD8α+ dendritic cells in the draining lymph node for cross-presentation
Authors: Harada Naozumi , Muraoka Daisuke , Tajima Kazuo , Shiku Hiroshi
Published: February 2011

Exogenous protein antigens per se poorly stimulate CD8+ T cells when used as immunogens. Hence, this could be a major drawback of cancer vaccines with protein antigens. To overcome the problem, we developed cholesteryl pullulan (CHP) as a delivery system for protein antigens. A series of in vitro and in vivo experiments and clinical studies confirmed that a therapeutic cancer vaccine containing the complex of CHP and protein antigen such as NY-ESO-1 or HER2 efficiently stimulated not only CD4+ T cells but also CD8+ T cells. To understand the mechanism underlying such effect of CHP, we investigated the tissue distribution of NY-ESO-1 protein complexed with CHP (CHP-NY-ESO-1) after its subcutaneous injection into mice by tracing the protein using NY-ESO-1-specific sandwich ELISA or FACS analysis with fluorescently labeled protein. By sandwich ELISA, NY-ESO-1 protein was detected only in the skin at the injection site, and its content was significantly higher with CHP-NY-ESO-1 than with NY-ESO-1 alone, indicating that CHP would make NY-ESO-1 protein antigen more persistent in vivo. In FACS analysis, fluorescence-positive cells which incorporated NY-ESO-1 protein were detected only in the draining lymph node among the lymphoid tissues such as draining/non-draining lymph nodes, spleen and peripheral blood mononuclear cells. Interestingly, the percentage of fluorescence-positive cells in the whole lymphocytes of this tissue was much higher with CHP-NY-ESO-1 (6.4%) than with NY-ESO-1 alone (0.9%). The lymphocytes capably incorporated fluorescence were CD11b+ cells and CD11c+ cells that include professional antigen-presenting cells such as macrophages and dendritic cells, respectively. In the mice received CHP-NY-ESO-1, the percentages of fluorescence-positive CD11b+ cells and CD11c+ cells were 53% and 66% at the highest, respectively, while these values in the mice with NY-ESO-1 alone were around 10%. Among the CD11c+ cells, as well as other dendritic cell subsets, the fluorescence was detected in the CD8α+ cells which are known to be capable to efficiently cross-present protein antigens. These results suggest that CHP would be an “immunotransporter” which efficiently delivers protein antigens to the antigen-presenting cells in draining lymph nodes. Hence, CHP may facilitate the presentation of protein antigens to both CD8+ T cells and CD4+ T 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 2408.
Abstract 2414: Comparable effect of p53 peptide vaccine in adjuvant or pulsed on dendritic cells in ovarian cancer patients: A gynecologic oncology group study
Authors: Rahma Osama , Achtar Ed , Czystowska Malgorzata , Szajnik Marta E. , Wieckowski Eva , Bernstein Sarah , Herrin Vincent E. , Steinberg Seth M. , Merino Maria , Gooding William , Visus Carmen , DeLeo Albert B. , Berzofsky Jay A. , Whiteside Theresa L. , Wolf Judith K. , Bell Jeffrey G. , Khleif Samir N.
Published: February 2011

Purpose: Peptide antigens have been administered by different approaches in cancer vaccine therapy, including direct injection or pulsed onto dendritic cells; however, the optimal delivery method is still debatable. In this study, we describe the immune response elicited by the wild-type (wt) p53 vaccine using two approaches.

Experimental Design: Twenty-one HLA-A2.1 patients with stage III, IV, or recurrent ovarian cancer overexpressing the p53 protein with no evidence of disease were treated in two cohorts. Arm A received subcutaneous wt p53:264-272 peptide admixed with Montanide and GM-CSF. Arm B received wt p53:264-272 peptide-pulsed dendritic cells intravenously. Interleukin-2 (IL-2) was administered in both cohorts in alternative cycles. The immunologic response was assessed by ELISPOT and tetramer assays.

Results: Nine of 13 patients (69%) in arm A and 5 of 6 patients (83%) in arm B had an immunologic response. The vaccine caused no serious systemic side effects. IL-2 administration resulted in grade 3 and 4 toxicities in both arms and directly induced expansion of T regulatory cells. The median overall survival was 40.8 and 29.6 months for arm A and B, respectively; the median progression-free survival was 4.2 and. 8.7 months, respectively.

Conclusion: We demonstrated that using either vaccination approach could generate comparable specific immune responses against the wt p53 peptide with minimal toxicity. Accordingly, our findings support the utilization of the less demanding subcutaneous method. Furthermore, as IL-2 may add toxicity and induce T regulatory cells, which can suppress the immune response, therefore, it may not be needed in future trials.

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 2414.
Abstract 2424: Synergistic induction of apoptosis in human solid cancer cells by Lexatumumab and cisplatin in a caspase-dependent manner
Authors: Xiuxian Wu , Inui Masashi , Sugimoto Mikio , Kakehi Yoshiyuki
Published: February 2011

Introduction and Objectives: Mapatumumab and Lexatumumab are human agonistic antibodies against TNF-related apoptosis-inducing ligand (TRAIL)-receptor 1 (TRAIL-R1) and TRAIL-R2, respectively, which induce apoptosis specifically in cancer cells. Here, we investigated whether chemotherapeutic agents enhance Mapatumumab- or Lexatumumab-mediated apoptosis against various solid cancer cells.

Methods: Cytotoxicity and synergy was assessed by MTT and isobolograpic analyses, respectively. TRAIL-R2 expression was detected by real-time RT-PCR, flow cytometry, and Western blotting. Apoptosis was assessed by DNA Ladder and caspase assays.

Results: Treatment of ACHN human renal cell carcinoma (RCC) cells with cisplatin combined with Mapatumumab did not overcome resistance to these agents. However, treatment with cisplatin in combination with Lexatumumab had a synergistic cytotoxicity. Synergy was also achieved in six primary RCC cell cultures. Lexatumumab and cisplatin also synergistically enhanced apoptosis. Pretreatment with cisplatin followed by Lexatumumab resulted in high cytotoxicity compare with the reverse sequence. Cisplatin significantly increased TRAIL-R2 expression at both mRNA and protein levels. Furthermore, the combination of Lexatumumab and cisplatin significantly enhanced caspase-8 activity, Bid cleavage, up-regulation of Bax, cytochrome c release, and caspase-9, caspase-6, and caspase-3 activities. Importantly, the activation of capase-8 was significantly abrogated by the specific inhibitors of caspase-9, caspase-6, and caspase-3. Furthermore, combination-induced cytotoxicity was significantly suppressed by DR5: Fc chimeric protein and the specific inhibitors of caspase-8, caspase-9, caspase-6, and caspase-3. Similar effect was observed in prostate cancer, bladder cancer, lung cancer, and cervical cancer cells.

Conclusions: Cisplatin sensitizes solid cancer cells to Lexatumumab-induced apoptosis by potentiation of the extrinsic and intrinsic apoptotic pathways that lead to enhancement of caspase activation, particularly caspase-8. These results suggest that combination of cisplatin plus Lexatumumab is promising against solid 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 2424.
Abstract 243: Characterization of CDK5RAP3 in hepatocellular carcinoma
Authors: Mak Grace W.Y. , Ng Irene O.L. , Ching Y.p.
Published: February 2011

CDK5 regulatory subunit associated protein 3 (CDK5RAP3, also called C53 and LZAP) is a novel cyclin-dependent kinase 5 (CDK5) activator interacting protein that has been shown to regulate apoptosis induced by genotoxic stress. More recently, CDK5RAP3 has also been found to regulate p53 and NF-κB signaling. Here, we have characterized the role of CDK5RAP3 in hepatocellular carcinoma (HCC) cell lines. Using real-time quantitative RT-PCR, we found that 58% (39/67) of human HCC samples had significantly higher expression (at least 2-fold) of CDK5RAP3 transcripts as compared to their corresponding nontumorous livers. The overexpression of CDK5RAP3 was statistically correlated with clinicopathological feature of more metastatic phenotype. Using stable expression clones, we demonstrated that knockdown of CDK5RAP3 in HCC cell line inhibited anchorage-independent growth and tumorigenicity of cells while overexpression of CDK5RAP3 remarkably promoted tumorigenicity of cells. Our western blotting data indicated that knockdown of CDK5RAP3 in HCC cell line SMMC-7721 resulted in an upregulation of p14ARF. Using luciferase reporter assay, we showed that ectopic expression of CDK5RAP3 suppressed p14ARF promoter activity in a dose-dependent manner. Taken together, our data provide the first evidence that CDK5RAP3 is overexpressed in HCC and overexpression of CDK5RAP3 may contribute to the development of HCC via downregulation of p14ARF. In summary, CDK5RAP3 is a potential oncoprotein in promoting hepatocarcinogenesis, the regulation behind may attribute to its regulation on p14ARF 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 243.
Abstract 2430: Gastric cancer cell lines with c-Met gene amplification are sensitive to growth inhibition by the bivalent c-Met antibody LA480
Authors: Tetreault Jonathan , Xia Jinqi , Zeng Wei , Wortinger Mark , Cunningham Jason , Feroze Farhana Mohammed , Yu Kun , Chow Chi-Kin , Lu Jirong , Denning Irene , Vaillancourt Peter , Davies Julian , Liu Ling
Published: February 2011

Gastric cancer is common in Asia, and advanced disease is typically incurable. Development of effective treatments for gastric cancer is thus a high priority to address this highly unmet clinical need. c-Met gene amplification resulting in c-Met overexpression and pathway activation has been highly linked to tumor progression and metastasis. It has been reported that approximately 10-20% of gastric tumors have increased copy numbers of the MET gene. c-Met- amplified gastric cancer cell lines that are dependent on an activated c-Met pathway for cell growth have been reported to be highly sensitive to growth inhibition by small molecule inhibitors of c-Met. We report that LA480, a humanized monoclonal antibody specific for c-Met, can directly inhibit HGF-independent in vitro cell proliferation of three c-Met- amplified Asian gastric cell lines (MKN45, SNU-5, and NUGC-4), in a dose-dependent manner. After 48 hours of treatment with LA480, 60-70% percent inhibition of 3H-thymidine incorporation was observed. For one of these gastric lines (NUGC-4), sensitivity to LA480 increases with continuous passage of the culture. This increased sensitivity to LA480 over time appears to be associated with increased expression of cell surface c-Met and increased constitutive phospho-Met expression. In addition to inhibiting proliferation of c-Met- amplified Asian gastric lines, LA480 also directly inhibited proliferation of a c-Met- amplified gastric cell line (Hs 746T) from a Caucasian patient. These data suggest that targeting c-Met amplification with LA480 may be a promising treatment for gastric cancer.

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 2430.
Abstract 2434: Novel primate-crossreactive BiTE antibodies that eliminate cancer cells expressing cMet, IGFR-1, FAP-alpha, PSCA, Endosialin, CAIX or Her2/neu
Authors: Raum Tobias J. , Rau Doris , Kischel Roman , Bluemel Claudia , Hausmann Susanne , Steiger Carola , Meier Petra , Ebert Evelyn , Mangold Susanne , Hoffmann Patrick , Strasser Susanne , Cierpka Ronny , Kvesic Majk , Baeuerle Patrick A. , Kufer Peter
Published: February 2011

BiTE antibodies are designed to transiently connect target cells with T cells of patients for induction of potent redirected lysis of cancer cells. Clinical proof-of-concept has been obtained with CD19/CD3-bispecific BiTE antibody blinatumomab (MT103), which showed a high response rate in patients with relapsed/refractory non-Hodgkin's lymphoma and acute B-lymphocytic leukemia. An EpCAM/CD3-bispecific BiTE antibody called MT110 currently is in a dose-escalating phase 1 study in patients with lung or gastrointestinal cancers, and BiTE antibodies targeting carcinoembryonal antigen (CEA), EGFR, CD33 or melanoma chondroitin sulfate proteoglycan (MCSP) are in earlier stages of development. Here, we have generated a series of novel BiTE antibodies for cancer therapy that are based on our novel BiTE antibody platform, which is human in sequence and cross-reactive between human and non-human primate antigens.

One set of target antigens selected for new BiTE generation is also targeted by conventional monoclonal antibodies that are in various stages of clinical development. These are mesenchymal-epithelial transition factor (cMet), insulin-like growth factor receptor type I (IGFR-1), prostate-specific stem cell antigen (PSCA), carboanhydrase type IX (CAIX) and Her2/neu. These targets are all expressed by cancer cells of solid tumors, and some also by their so-called cancer stem cells. By targeting fibroblast activating protein alpha (FAP-alpha), a first BiTE antibody was generated that not only targets sarcoma cells but also stromal fibroblasts, which constitute the stromal tissue of many human cancers. By targeting endosialin (CD248), a first BiTE antibody was generated targeting cancer blood vasculature.

We show that various single-chain antibodies specific for the seven target antigens cMet, IGFR-1, FAP-alpha, PSCA, CAIX, Her2/neu and endosialin could in each case be used to construct novel BiTE antibodies. All BiTE antibodies showed bispecific binding, cross-reactivity between human and Cynomolgus monkey antigens, and, most importantly, potent redirected lysis of CHO cells expressing the respective target antigen. This shows that new T cell-engaging BiTE antibodies can be made specific for a great variety of potential tumor-associated and also for stromal antigens.

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 2434.
Abstract 2435: Validation of Cynomolgus monkeys as relevant species for safety assessment of a novel human BiTE antibody platform for cancer therapy
Authors: Rattel Benno , Kischel Roman , Thomas Oliver , Friedrich Matthias , Rau Doris , Ebert Evelyn , Raum Tobias , Wolf Andreas , Mangold Susanne , Kvesic Majk , Baeuerle Patrick A. , Kufer Peter
Published: February 2011

Nonclinical safety assessment of biopharmaceuticals for human use relies on their cross-reactivity with the respective target in other species, the absence of which precludes testing in commonly used animal models. This is the case for a first-generation of T cell-engaging BiTE antibodies, which solely cross-react with T cells of chimpanzee. BiTE antibodies are designed to transiently connect the CD3 subunit of T cell receptors on T cells with an antigen on target cells. This can very effectively activate T cells, leading to a potent redirected lysis of target cells.

Here, we have generated a series of novel human anti-CD3 antibodies that cross-react with non-human primate species, including Cynomolgus monkeys (Macaca fascicularis) and marmoset (Callithrix jacchus). The variable domains of a selected anti-CD3 antibody were used to generate next-generation BiTE antibodies specific for (1) CD33, a target antigen for treatment of acute myelogenous leukemia, (2) melanoma-associated chondroitin sulfate proteoglycan (MCSP), a potential target for treatment of melanoma, and (3) epidermal growth factor receptor 1 (EGFR), a validated target for treatment of colorectal cancer.

In vitro, CD33-, MCSP- and EGFR-specific BiTE antibodies showed comparable binding to CD3 on human and macaque T cells, and to CHO cells expressing CD33, MCSP and EGFR of human or macaque origin. In human and macaque cell-based assays, bispecific binding triggered redirected target cell lysis as well as conditional T cell activation, as previously described for first-generation BiTE antibodies.

When continuously infused for one week to Cynomolgus monkeys, the CD33-specific BiTE antibody caused depletion of CD33-expressing peripheral blood cells at tolerated doses, and reached a maximum tolerated dose (MTD) at 120 µg/m2/day. The EGFR-specific BiTE antibody reached an MTD at 150 µg/m2/kg. In contrast, the MCSP-specific BiTE antibody was well-tolerated up to a dose of 5,000 µg/m2/day. Our primate data suggest that the tolerability and toxicity of CD33-, MCSP- and EGFR-specific BiTE antibodies were apparently determined by target cell-dependent bispecific binding, and not by monovalent binding to CD3 on macaque T cells. Our data validate the Cynomolgus monkey as a relevant species for assessing the safety and tolerability of next-generation BiTE antibodies that use a more broadly cross-reactive anti-CD3 human single-chain antibody.

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 2435.
Abstract 2441: An innovative treatment modality of antibody therapy against intracellular tumor antigens
Authors: Noguchi Takuro , Kato Takuma , Muraoka Daisuke , Ikeda Hiroaki , Kondo Satoshi , Ritter Gerd , Old Lloyd J. , Shiku Hiroshi , Nishikawa Hiroyoshi
Published: February 2011

Therapy with mAb directed against cell surface antigens of cancer is now a well-established treatment modality for human cancer. However, many tumor antigens of great interest have an intracellular location, and therefore not considered suitable targets for antibody therapy. On the other hand, it has been reported that mAbs targeted against specific tumor antigens induce cellular immune response including CD8+ T cells. This raises the possibility that the anti-tumor effects of mAb therapy may partially depend on enhanced cross-priming mediated by APCs following the uptake of antigen released from tumors complexed with Ab (immune-complex). Here, we investigated whether mAb therapy could be applicable to intracellular antigens using a transplantable colon carcinoma CT-26 expressing NY-ESO-1 (a well-studied human cancer/testis antigen), an intracellular antigen lacking cell surface expression. Immune responses to NY-ESO-1 in the murine model closely parallel NY-ESO-1 humoral and cellular immune responses in humans. To accentuate the natural release of intracellular NY-ESO-1 from dying tumor cells to facilitate immune-complex formation, we employed the effect the anti-cancer drug on the response to NY-ESO-1 mAb.

The combination of NY-ESO-1 mAb and 5-fluorouracil exhibited a strong and long-lasting anti-tumor effect that could not be achieved by treatment with either 5-fluorouracil or mAb alone. The anti-tumor effect induced by combination therapy was no longer observed when Fc-depleted Fab antibodies were used, indicating a Fc-dependent action on APCs. Furthermore, augmented anti-tumor effects observed by the combination therapy was associated with strong NY-ESO-1-specific CD8+ T cells in draining lymph nodes and tumor sites, and upregulation of maturation markers on dendritic cells. The opsoniziation of released intracellular tumor antigens with injected mAb facilitates uptake by APC and presentation to CD8+ T cells, leading to accelerated and augmented active immunization. The data indicate that mAb to intracellular molecules of cancer represents a promising approach to cancer immunotherapy.

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 2441.
Abstract 245: An oncogenic role for CCAAT/enhancer binding protein beta in non-small cell lung cancer
Authors: Thoennissen Nils H. , Iwanski Gabriela B. , Ji Jianfei , Ikezoe Takayuki , Lee Kunik , Adachi Yoshihiro , Takeuchi Tamotsu , Furihata Mutsuo , Park Dorothy J. , Gombart Adrian F. , Koeffler H. Phillip
Published: February 2011

The transcription factor CCAAT/enhancer binding protein (C/EBP) beta is known to be a mediator of cellular transformation and tumorigenesis in human colon, breast and ovary cancer. In this study, we focused on C/EBPbeta and its potential role in non-small cell lung cancer (NSCLC). Immunohistochemical staining of 60 human NSCLC samples (adeno- (31), squamous cell - (25), small cell - (1), large cell - (2), and adeno-squamous cell (1) carcinoma) showed that more than 50% of the tumor samples were significantly positive for nuclear staining of C/EBPbeta compared to healthy airway epithelial cells. Retrovirus-delivered siRNA knock-down of C/EBPbeta expression in human NSCLC cell lines A549, H520, and H460 markedly reduced clonogenic growth, which was accompanied by a significant reduction in cyclooxygenase(COX)-2 expression, increase in G0/G1 cells, and PARP cleavage activity. In contrast, using NSCLC cell line Calu-3 with a moderate intrinsic level of C/EBPbeta, forced expression of this transcription factor even increased clonogenic growth and cell proliferation. Whereas recent studies suggested that family member C/EBPalpha has growth-inhibitory properties in airway epithelial cells, our present data indicate an oncogenic role of C/EBPbeta in NSCLC and may represent a new target for cancer co-therapy to increase response to existing chemotherapeutic regimens.

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 245.
Abstract 2455: The fjord region (±)-anti-dibenzo[a,l]pyrene-11,12-dihydrodiol-13,14-epoxides ((±)-anti-DB[a,l]PDE), is a powerful and specific inducer of squamous cell carcinoma in the oral cavity of mice
Authors: Chen Kun-Ming , Guttenplan Joseph B. , Aliaga Cesar , Cooper Timothy , Deltondo Joseph , Kosinska Wieslawa , Sharma Arun K. , Sun Yuan-Wan , Zhang Shangmin , Jiang Kun , Bruggeman Richard , Amin Shantu , El-Bayoumy Karam
Published: February 2011

Head and neck squamous cell carcinoma (HNSCC), the sixth most common malignancy, represents a major international public health problem. The vast majority of HNSCC occurs in the oral cavity. Cancer of the mouth strikes about 30,000 Americans each year and less than 50% of patients survive 5 years after diagnosis. Tobacco and alcohol use are considered major causative agents. Current preclinical animal models to study oral carcinogenesis do not accurately mimic the biology of this disease in humans. We have developed a new mouse model by the application of the fjord region diol epoxide, (±)-anti-DB[a,l]PDE, a metabolite of the tobacco constituent dibenzo[a,l]pyrene (DB[a,l]P), to the oral cavity, and we showed for the first time that it is a powerful and specific inducer of oral squamous cell carcinoma (OSCC) in both tongue and oral tissues. Briefly, two groups of B6C3F1 mice (20/group) received doses of (±)-anti-DB[a,l]PDE (6, and 3 nmol) administered into the oral cavity; additional groups received the vehicle or left untreated as controls, 3 time a week for 38 weeks. Mice were sacrificed one year after the first carcinogen administration. The high dose induced 74 % and 100 % OSCC in the tongue and oral tissues, respectively; the corresponding values at the lower dose were 45 % and 89 %. Consistent with the carcinogenicity, we demonstrated powerful mutagenicity in CII gene in big blue mice and the mutation spectra in both tongue and oral tissues revealed similarity in the profiles between DB[a,l]PDE, and that of p53 mutations found in human OSCC. Using immunohistochemistry, we showed that (±)-anti-DB[a,l]PDE resulted in overexpression of p53 protein in malignant tissues compared to normal oral tissues and tongues.

Collectively, we developed a non-surgical whole animal model which is the first to demonstrate the remarkable carcinogenicity and specificity of (±)-anti-DB[a,l]PDE in the oral cavity; this model is an appropriate platform for further exploring the genetic and epigenetic alterations that can truly account for the development of OSCC on humans. In addition, this model can be employed to study efficacies of novel cancer chemopreventive agents.

Supported by NCI grant no. R01 CA 100924.

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 2455.