bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2355: Role of secreted frizzled-related protein 3 (sFRP3) in human renal cell carcinoma
Authors: Hirata Hiroshi , Hinoda Yuji , Ueno Koji , Majid Shahana , Saini Sharanjot , Dahiya Rajvir
Published: February 2011

Wnt/beta-catenin signaling is involved in renal cancer. Among Wnt antagonist families, sFRP3 is generally thought to be an inhibitor of Wnt signaling in several cancers. Here we reported the functional significance of sFRP3 in renal cell carcinoma. We performed immunohistochemistry on kidney tissue microarray and confirmed the expression level of sFRP3 in normal kidney and renal cancer cell lines (primary and metastatic). We transfected sFRP3 to the primary renal cancer cell lines and performed several functional analysis (MTS, colony formation, apoptosis, cell cycle, invasion, angiogenesis, and in vivo study). We also knocked down sFRP3 mRNA in metastatic cell lines using siRNA technique and performed several functional analysis. We found the level of sFRP3 protein was high in normal kidney, low in primary renal cancer tissues and high in metastatic renal cancer tissues. We performed dual sFRP3 mRNA expression experiments to determine the role of sFRP3 using primary and metastatic renal cancer cell lines. Functional analysis showed increased numbers of viable and invaded cells, tube formation and decreased numbers of apoptotic cells in sFRP3 transfected A498 cells. Promotion of tumor growth was also observed in nude mice injected with sFRP3 transfected A498 cells. In contrast, the number of viable cells and invasive cells was decreased in sFRP3 mRNA knocked down metastatic cells (ACHN and Hs891.T). To investigate the mechanism of sFRP3 function, we performed microarray analysis to see which genes were up or down regulated by sFRP3 expression. Among these genes, MMP-3 and ANGPT1 were significantly up-regulated in sFRP3 transfected cells. This is the first report to show that sFRP3 expression promotes cell growth, invasion, and inhibition of apoptosis in renal cancer cells. Furthermore, our results suggest that sFRP3 may play a role in invasion and angiogenesis by enhancing MMP-3 and ANGPT1 expression. The changes in sFRP3 expression levels in normal, primary cancer, and metastatic renal cancer tissues suggest that sFRP3 may play dual roles as a tumor suppressor gene and an oncogene during renal cancer progression.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2355.
Abstract 2360: Cell size and shear stress resistance do not correlate with metastatic efficiency
Authors: Barnes James M. , Henry Michael D.
Published: February 2011

Metastasis is the leading cause of death in patients with epithelial cancers. It has been observed both clinically and experimentally that metastasis is a very inefficient process. It is believed that the majority of primary cancer cells entering the bloodstream are cleared rapidly, likely by destruction under hemodynamic shear forces, thus greatly reducing the number of surviving cells which can go on to colonize a secondary site. It would thus seem advantageous for metastatic cancer cells to adapt mechanisms to resist this type of stress. One factor which may influence shear stress susceptibility is cell size; the larger a cell is the more shear stress this cell will experience while in circulation. Here, we report the cell size and shear stress analysis of a panel of well described cancer cell lines and their metastatic derivatives obtained from metastatic lesions in mice. We found no correlation between the ability of a cancer cell to metastasize with a reduction in cell size or an increase in shear force resistance. Our data suggest that the loss of tumor cells by hemodynamic shear force is lower than often estimated. We conclude that the metastatic cascade does not generally select for cells of a smaller size and that shear stress resistance does not predict the aggressiveness of a particular cancer cell line. These findings support the hypothesis that the later steps in metastasis, such as extravasation and colonization, dictate the inefficiency of this process. Interestingly, although metastatic derivatives did not tend to exhibit increased resistance to shear force, there were certainly differences between cancer cell lines of different tissue origin. This fact may become important when designing isolation methods for circulating tumor cells, the analysis of these studies, and the prognostic value of quantifying circulating 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 2360.
Abstract 2361: Fractalkine and CX3CR1 are involved in the early stages of skeletal metastasis
Authors: Jamieson Whitney L. , Dorgham Karim , Deterre Philippe , Fatatis Alessandro
Published: February 2011

It is estimated that eighty percent of breast cancer (BCa) and ninety percent of prostate cancer (PCa) patients develop late-stage skeletal metastases. Metastatic disease is the major cause of death in these patients and only palliative treatments for clinically established skeletal metastases are available. In order to develop effective preventive and curative therapies, it is essential that mechanisms involved in cancer cell bone colonization are fully understood. Our previous in vitro experiments have shown that the chemokine fractalkine (FKN, CX3CL1) and its receptor, CX3CR1, mediate adhesion of PCa cells to bone marrow endothelial cells as well as their migration toward osteoblasts. We have also shown that CX3CR1 is highly expressed in BCa and PCa cell lines as well as ex vivo prostate and mammary human cancer tissues, whereas FKN is expressed by stromal cells of the bone marrow. Therefore, we hypothesize that FKN and CX3CR1 are involved in the arrival and arrest of cancer cells to the bone during the metastatic process. To test our hypothesis, we employed a mouse model of metastasis in which fluorescent human cancer cells are inoculated in the left cardiac ventricle. Using a histology-stereomicroscopy combination approach, we detect single cancer cells in the bone microenvironment as early as 24 hours after their delivery into the blood stream. Analyses at later time-points also reveal the progression of small cancer foci into macroscopic metastases. Inoculation of PCa or BCa cells in FKN-null mice shows a significant decrease in the number of cancer cells disseminated to the skeleton by hematogenous route. In parallel experiments, exogenous over-expression of CX3CR1 in BCa cells increases their arrival to bone. On the other hand, upon expression of a CX3CR1 functional mutant - unable to mediate adhesion to FKN - the dissemination of BCa cells to the skeleton is significantly impaired. Finally, we used a newly engineered inhibitor of CX3CR1 to counteract skeletal metastases in vivo.

In conclusion, our data present strong evidence that the FKN/CX3CR1 pair is involved in the early stages of prostate and breast cancer bone metastasis. Thus, altering the interactions between FKN and its receptor represents an attractive therapeutic approach for the prevention of skeletal metastases from prostate and breast 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 2361.
Abstract 2374: Integrin-linked kinase (ILK) promotes melanoma angiogenesis by activating IL-6 signaling pathway
Authors: Wani Aijaz A. , Li Gang
Published: February 2011

Integrin-linked kinase (ILK) is a highly conserved serine-threonine protein kinase involved in cell-ECM interactions, cytoskeletal organization, and cell signaling. Overexpression of ILK in epithelial cells leads to anchorage-independent growth with increased cell cycle progression. Previously, we have shown that ILK upregulation strongly correlates with melanoma progression, invasion and inversely correlates with 5-year survival of melanoma patients. We also found that ILK knockdown impeded melanoma cell migration, invasion and impaired the growth of melanoma xenografts in severe combined immunodeficient (SCID) mice. However, the molecular mechanism by which ILK enhances melanoma angiogenesis is currently unknown. In the present study, we found that pro-angiogenic molecule interleukin-6 (IL-6) is the downstream transcriptional target of ILK in melanoma cells. ILK overexpression increased IL-6 whereas shRNA mediated silencing of ILK suppressed IL-6 expression both transcriptionally and translationally. By electromobility shift assays, we found that ILK enhanced the binding of transcription factor (NF-κB) to IL-6 promoter and this binding was reduced in ILK knockdown cells. Furthermore, Conditioned media (CM) from ILK- overexpressing cells enhanced the tube forming ability of HUVEC cells in vitro. This tube formation by HUVEC cells was IL-6 dependent as CM from IL6-siRNA treated ILK-overexpressing melanoma cells inhibited the tube formation of HUVEC cells. In order to delineate the mechanism by which ILK-upregulated IL-6 can enhance the growth of endothelial cells, further analysis of the downstream targets of IL-6 signaling showed an increase in activity and nuclear translocation of STAT3 in ILK-overexpressing cells. As STAT3 binds to VEGF promoter, we found that VEGF levels were elevated in ILK-overexpressing cells and declined in ILK knockdown cells, suggesting that ILK may regulate VEGF expression through IL-6 pathway by upregulating and activating STAT3.

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 2374.
Abstract 2378: Involvement of leptin-activated canonic and non-canonic kinases in VEGF regulation in breast cancer
Authors: Xu Yanbo , Watters Amber , Zhang Yuanyuan , Gillespie Corey , Zhou Weiqiang , Guo Shanchun , Leibovich Samuel J. , Gonzalez-Perez Ruben Rene
Published: February 2011

We have previously reported that in vitro and in vivo leptin signaling up-regulates VEGF in mouse and human mammary tumor cells (MT) but the specific molecular mechanisms involved are largely unknown. Here we describe how leptin activated canonic and non-canonic signaling pathways to induce specific transcription factors (TF) essential for the transcriptional activation of VEGF gene in mouse MT (4T1, EMT6 and MMT). Basal expression of VEGF/VEGFR2, leptin/OB-R and ER-α was initially investigated in MT. Leptin dose-response and time-course effects on VEGF levels were determined by ELISA. Several pharmacological inhibitors for key leptin-target kinases and TF were used to dissect the signaling pathways involved in leptin-induced levels of VEGF protein and mRNA in MT. A series of VEGF-promoter Luc-reporters [full-length & transcription factor (TF)-binding deletions: HRE, AP1, AP2, NFκB and SP1] were transfected into MT to analyze leptin regulation of VEGF transcription. Our data show that MAPK, PI-3K (canonic) and PKC, JNK and p38 MAP kinase (non-canonic) signaling pathways were generally involved in leptin regulation of VEGF. Analysis of cis-acting elements of VEGF promoter shows that leptin activation of HIF-1α is essential for transcription in all MT. This was mainly linked to MAPK, PI-3K, PKC, JNK and p38 signaling. Leptin-induced MAPK, JNK and p38 to activate NFκB that was essential for VEGF expression in high producer MT (EMT6 and MMT). Leptin through all canonic signaling pathways activates SP1 required for VEGF regulation in 4T1 cells. In contrast, AP1 was not involved but AP2 repressed leptin-mediated transcription of VEGF. Overall, these data suggest that leptin signaling regulates VEGF gene in MT cells mainly through HIF-1α and NFκB. These results delineate for first time the mechanisms for leptin regulation of VEGF and, reinforce the idea that the disruption of leptin signaling could impact breast cancer growth by inhibiting MT proliferation and angiogenesis. This work was supported in part by NIH/NCI 1SC1CA138658-02; NIH/UAB Breast SPORE Career Development Award; BC 504370 Susan G. Komen Foundation for the Cure and the Georgia Cancer Coalition Distinguished Cancer Scholar Award (to RRGP).

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 2378.
Abstract 2385: Evidence and actions for nuclear localization of vascular endothelial growth factor receptor 1 (VEGFR1) in endothelial and cancer cells
Authors: Wu Li-Wha
Published: February 2011

Abstract:

Vascular endothelial growth factor receptor 1 (VEGFR1) is a receptor tyrosine kinase (RTK) predominantly expressed in endothelial cells. Recent studies have shown that VEGFR1 is also expressed in several types of human cancer cells, and its expression correlates with cancer progression. Moreover, higher VEGFR1 expression worsened the outcome of lung cancers, breast cancer, and pancreatic cancer patients following cancer treatment. A new mode of signal transduction mediated by RTK is through internationalization of ligand-bound receptor rather than through activation of intracellular signaling cascades by the interaction of ligand and receptor on the cell surface. Epidermal growth factor receptor is the prototype for such new mode of action. One previous study has shown that VEGFR1 was colocalized with nuclear STAT1 in chronic lymphocytic leukemia B cells treated with VEGF-A, indicating a possible role for VEGFR-1 in the nucleus. Consistent with the notion, we found that the intracellular domain of VEGFR1 behaved as a transcriptional activator in yeast two-hybrid system. Second, VEGFR1 could shuttle between cytosol and nucleus following VEGF-A treatment in endothelial cells. Third, nuclear and cytosolic fractionation indicated the presence of VEGFR1 in the nuclear compartment of oral cancer and human endothelial cells. We thus hypothesized that VEGFR1 might harbor a nuclear localization signal (NLS) in the protein. Using a cytosolic protein pyruvate kinase fused with green fluorescent protein (GFP) and the putative NLS derived from VEGFR1 together with serial deletions and fluorescence microscopy, we were able to alter the subcellular location of pyruvate kinase fusion protein. Moreover, mutations of crucial arginine or lysine residues in the putative NLS significantly reduced the nuclear presence of VEGFR1 in the transfected cells. More studies are needed to address the molecules involved in nuclear transport of VEGFR1 and the target DNA sequence recognized by nuclear VEGFR1. The functional consequence of VEGFR1 localization in different subcellular compartments will be also studied by the use of oral cancer cells as a model.

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 2385.
Abstract 2386: High-throughput assessment of the VEGFR2 kinase activity profile in pediatric brain tumor tissue
Authors: Sikkema Arend H. , de Bont Eveline S. , Molema Grietje , Dimberg Anna , Zwiers Peter J. , Diks Sander H. , Hoving Eelco W. , Kamps Willem A. , Peppelenbosch Maikel P. , den Dunnen Wilfred F.
Published: February 2011

For enhanced tumor cell survival and progression, tumors depend on angiogenesis. Vascular endothelial growth factor receptor (VEGFR) signaling plays a major part in this process. Previously we generated a comprehensive description of the biological tyrosine kinase activity present in pediatric brain tumor tissue lysates by the application of a peptide microarray containing 144 different tyrosine kinase peptide substrates (Sikkema et al., Cancer Res 2009). The results suggested the presence of activated VEGF receptors in pediatric pilocytic astrocytoma tissue. We hypothesized that this detected VEGFR activity reflects the angiogenic status of the tumor endothelium.

In this study we demonstrated that VEGFR2 expression is not present in a pilocytic astrocytoma cell line. Moreover, proximity ligation assays on tumor cryosections showed VEGFR2 phosphorylation, primarily localized on endothelial cells. qRT-PCR analysis of endothelial markers and VEGFRs showed a 10 to 30-fold enrichment of VEGFR2 expression on laser microdissected endothelium compared to whole tumor. Hence, we conclude that the endothelial cells are the high producers of VEGFR2 kinase activity in these tumors and therefore responsible for the VEGFR2 kinase activity that we observed in our kinase activity screens.

In conclusion, potential VEGFR kinase substrates present on the peptide microarray are adequately sensitive to pick up enzymatic activity which is present in only a subset of the total cell population within solid tumors such as pilocytic astrocytoma. In the near future, high throughput measurement of enzymatic activity of key players in angiogenesis could make a rapid assessment of the angiogenic profile of a tumor possible. This could serve as an indicator of anti-angiogenic treatment response and result in more personalized anti-angiogenic cancer treatment strategies.

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 2386.
Abstract 2406: Increased efficacy of combination of IL-13 receptor-targeted cytotoxin and DNA vaccine in murine tumor models
Authors: Nakashima Hideyuki , Husain Syed R. , Puri Raj K.
Published: February 2011

Recent studies show that interleukin-13 receptor α2 chain (IL-13Rα2) is a tumor-associated antigen that is a promising potential target for immunotherapy. High levels of IL-13Rα2 are expressed on a variety of human tumors, but its precise role in tumor immunology is not known. IL13-PE, a targeted cytotoxin composed of IL-13 fused to mutated form of Pseudomonas exotoxin, induces specific killing of tumor cells expressing abundant levels of IL-13Rα2. In this study, we sought to determine the effect of IL13-PE in combination with DNA vaccination in murine MCA304 sarcoma and 4T1 breast carcinoma, which naturally over-express IL-13Rα2 antigen.

Three intramuscular prophylactic IL-13Rα2 DNA immunizations in syngeneic mice (n=6) on −8, −4, and −2 weeks resulted in partial protection (382 mm3) against subcutaneous MCA304 tumor challenge compared to mock vector vaccine (1498 mm3) on day 21. Three additional intratumoral (i.t.) treatments of IL13-PE on day 4, 6 and 8 post-tumor challenge further decreased tumor burden (85 mm3) by 78%, measured on the same day (day 21) compared to DNA vaccination alone. In an established MCA304 tumor model, mice receiving three i.t. injections of IL13-PE (on day 7, 9, and 11) and four subsequent DNA vaccinations (on day 13, 18, 23 and 28) reduced tumor size to 44 mm3 (94% less) compared to DNA vaccination alone (693 mm3) on day 24. For comparison, the untreated control tumors had a size of 1579 mm3 on day 24. Interestingly, the combination therapy with IL13-PE and DNA vaccination produced 50% complete responders; however, tumors in remaining 50% partial responders grew slowly. Combination therapy prolonged the mean survival to 60 days compared to control (24 days) and DNA only vaccinated groups (36 days). Similar results were also observed in the 4T1 murine breast carcinoma model. Combination therapy group showed higher CTL activity and IFN-γ release from splenocytes compared to the single therapy groups in both tumor models. Immunohistochemical analysis showed the infiltration of CD4 and CD8 positive T cells and expression of IFN-γ-induced chemokines (CXCL9 and CXCL10) in tumor tissues of immunized mice. These results suggest that combination therapy with IL13-PE and IL-13Rα2 DNA vaccination inhibited tumor growth in a T cell dependent manner. In addition to the combination therapy strategy, elimination of immunosuppressive elements such as regulatory T cells and myeloid-derived suppressor cells may further enhance tumor immunity.

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 2406.
Abstract 2432: Fully human anti-interleukin-6 (IL-6) monoclonal antibody suppresses the growth of human tumor xenograft in vivo
Authors: Zhong Haihong
Published: February 2011

Interleukin-6 (IL-6) is a pleiotropic cytokine that plays a major role in the response to injury or infection and is involved in the immune response, inflammation, and hematopoiesis. Aberrant expression of IL-6 has been implicated in many human diseases. In cancer, high levels of circulating IL-6 are observed in many tumor types and associated with poor outcome. We hypothesized that a monoclonal antibody that neutralizes IL-6 activity may be effective in inhibiting the progression of cancer. A fully humanized monoclonal antibody against human IL-6 (CAT-6001) was isolated by screening phage-display libraries. CAT-6001 completely neutralizes IL-6 activities via binding to the IL-6 receptor and signal transducer gp130 binding sites of human IL-6. We demonstrate that several cancer cell lines secrete high levels of IL-6 and soluble IL-6 receptor, and express gp130. High constitutive pStat3 activation is seen in several of these cell lines suggesting an IL-6 autocrine loop. Treatment of these cell lines with CAT-6001 effectively blocked phosphorylation of Stat3 and in some cell lines, functionally inhibited IL-6 induced cell proliferation. In vivo, CAT-6001 inhibited the growth of DU145, a human prostate cancer xenograft model, and significantly blocked the release of human IL-6 and inhibited Stat3 phosphorylation. Taken together, our data suggest that IL-6 signaling contributes to tumor growth, and provide support for the further development of CAT-6001 for the treatment of solid tumors.

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 2432.
Abstract 2474: Increased PKC expression in diacylglycerol-induced mammary carcinogenesis in the human c-Ha-ras proto-oncogene transgenic rat
Authors: Suzui Masumi , Yoshida Ichiro , Ishihara Masashi , Nakashima Makoto , Matsunaga Toshiyuki , Hara Akira , Fukamachi Katsumi , Tusda Hiroyuki
Published: February 2011

Protein kinase C (PKC) is implicated in cell proliferation and tumor promotion. PKC is activated by 1, 2-diacylglycerol (1, 2-DAG). An edible oil that includes 70% 1,3-DAG and 30% 1,2-DAG is commercially available in Japan, USA, and European countries. To examine whether the DAG oil enhances mammary carcinogenesis and to measure whether DAG affects the levels of PKC expression, we carried out this study with human c-Ha-ras proto-oncogene transgenic (Hras128) rats that are highly susceptible to mammary carcinogenesis. Hras128 rats (6-week-old) were randomly assigned into four experimental groups. DAG oil was administered to the rat as oral drops. Group 1 (G1): 0.5 mL triacylglyceraol (TAG) x2/week, Group 2 (G2): 0.5 mL DAG x2/week, Group 3 (G3): 0.5 mL TAG x1/week plus 0.5 mL DAG x1/week, Group 4 (G4): 0.5 mL DAG x1/2 weeks + 0.5 mL TAG x3/2 weeks. Experiment was terminated after 15 weeks. Mammary tumors were histologically identified as adenocarcinoma. Tumor incidence of G2 (77%) and G3 (77%) was significantly higher than G1 (22%). Tumor multiplicity of G2 (1.3 ± 1.1) was significantly higher than the other 3 groups (G1, 0.4 ± 1.0; G3, 0.9 ± 0.6; G4, 0.7 ± 1.0). In tumor samples, the mRNA expression levels of 6 PKC isoforms (α, δ, ε, η, λ, ν) increased compared to the adjacent normal tissues. In a parallel study, we also examined the levels of PKC expression in normal mammary tissues of Hras128 rats treated with either TAG or DAG oils alone for 4 weeks. The mRNA expression levels of 5 PKC isoforms (δ, ε, η, λ, θ) significantly increased in the DAG-treated normal tissues compared to the TAG-treated normal tissues. These findings provide evidence that treatment of Hras128 rats with DAG oil may promote mammary carcinogenesis by inducing expression of several PKC isoforms.

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 2474.
Abstract 2491: Biphasic effect of psoralidin on cell cycle regulatory markers in hormone refractory prostate cancer
Authors: Koduru Srinivas , Rohr Jurgen , Damodaran Chendil
Published: February 2011

Cell cycle deregulation has been strongly associated with the pathogenesis of prostate cancer (PCa). Overexpression of cell cycle regulatory proteins (cyclin D1-25%, cyclin A-35% and cyclin B-75%) have been reported in clinical PCa samples when compared to normal prostate tissue. Based on this, we rationalized that downregulation of cell cycle regulatory molecules may play an important role in chemoprevention/therapy of PCa. We previously published that psoralidin; a natural compound effectively suppresses PCa growth both in vitro and in vivo. Hence, in this study we determined whether psoralidin alters cell cycle regulatory proteins in PCa cells. To determine this we performed cell cycle analysis using PI staining and also studied the expression patterns and activity of various cell cycle regulatory markers using Western blot analysis and kinase assays respectively in two hormone refractory prostate cancer (HRPC) cells (PC-3 and DU-145) following treatment with or without psoralidin in dose dependent manner. Psoralidin has biphasic effect on PCa cells where at a lower concentration (40µM) caused a G0/G1 cell cycle arrest whereas at a higher concentration (60µM) caused a G2/M cell cycle arrest. Psoralidin at lower concentration induced expression of cyclin dependent kinase inhibitors (p27, p57, p21, p16) in PC-3 and DU-145 cells whereas at higher concentration upregulation of p18 was observed in both HRPC cells. Additionally, we found that at lower concentration cyclins (E & E2 and H) and cdks (6 and 9) are downregulated whereas at higher concentration psoralidin downregulated expression of cyclins (D1, A, E & E2, B1, D3 and H) and cdks (6, 7, 2 and 9) in both HRPC cells. At lower concentration psoralidin inhibits expression of cdc-2 and cdc-25B whereas at higher concentration cdc-2, −25A and −25B are downregulated in both PC-3 and DU-145 cells. Also, psoralidin inhibited phosphorylation of Rb at both lower and higher concentration in HRPC cells causing irreversible cell cycle arrest resulting in the induction of apoptosis. These results clearly highlight the potential of psoralidin as a therapeutic agent for PCa. Currently, we are investigating the effect of psoralidin on cell cycle markers using both xenograft and TRAMP models. The outcome of our study may enable bringing psoralidin to the mainstream of medicine for prevention and/or therapy for PCa.

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 2491.
Abstract 2563: Upregulation of miR-155 is associated with cisplatin resistance in the head and neck cancer UMSCC-10B/15S cells
Authors: Gupta Ashim , Zhu Shoumin , Sachdeva Mohit , Rao Krishna , Robbins Thomas , Mo Yin Yuan
Published: February 2011

Introduction: Cisplatin combination chemotherapy is the cornerstone of treatment for many cancers including head and neck cancer. These complexes bind to the DNA and form adducts such that they ultimately trigger apoptosis. Although initial platinum responsiveness is usually high, the majority of cancer patients will eventually relapse with cisplatin-resistant disease. Thus, chemotherapy resistance is a major clinical obstacle in the successful treatment of head and neck cancer. This study aims to investigate the role of microRNAs (miRNAs) in cisplatin resistance and to better understand the underlying mechanism of cisplatin resistance in the head and neck cancer UMSCC-10B/15S cells.

Methods: To determine whether aberrant miRNA expression is associated with cisplatin resistance in human head and neck cancer cells, we profiled miRNA expression in the cisplatin resistant UMSCC-10b/15S and the parental line UMSCC-10B cells by real-time PCR. Luciferase reporter assays and western blot were used to confirm targets for miR-155. We also suppressed miR-155 by anti-sense locked nucleic acid (LNA) oligo to further determine the role of miR-155 in cisplatin resistance.

Results: We showed that several miRNAs were altered in UMSCC-10B/15S and among them, miR-155 was the most significantly upregulated miRNA. This was also confirmed by in situ hybridization. Furthermore, ectopic expression of miR-155 was able to increase the resistance of UMSCC-10B cells to cisplatin, which is likely in part through targeting DMTF1, a tumor suppressor that directly binds to the Arf promoter to activate its expression, thereby inducing p53-dependent cell cycle arrest and apoptosis. To determine whether miR-155 directly targets DMTF1, we cloned 1.2 kb 3′-untranslated region (UTR) of DMTF1 into a luciferase reporter and demonstrated that miR-155 was able to suppress luciferase activity by over 70%. We also showed that the endogenous DMFT1 protein was reduced by over 50% in the miR-155 highly expressed resistant cells compared to the parental sensitive cells. Experiments are under way to determine whether ectopic expression of DMTF1 or its suppression by RNAi impacts cisplatin resistance in these cells.

Conclusion: These findings suggest that deregulation of miRNAs is in resistant head and neck cancer cells and miR-155 may play a causal role in cisplatin resistance through targeting the DMTF1 gene. As a result, suppression of miR-155 may provide a strategy to overcome the resistance in head and neck 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 2563.
Abstract 2567: Expression of BRAF splice variants lacking the catalytic domain predicts dacarbazine response in advanced melanoma
Authors: Busch Christian , Geisler Jürgen , Lillehaug Johan Richard , Lønning Per Eystein
Published: February 2011

BACKGROUND: Metastatic melanomas respond poorly to chemotherapy. The aim of this single institution study was to correlate BRAF and NRAS mutation status to dacarbazine (DTIC) response and outcome in metastatic melanomas.

METHODS: Snap-frozen tissue samples were obtained from 85 malignant melanoma deposits. Seventy-five of these were from patients evaluable for response to dacarbazine treatment, and BRAF and NRAS status was correlated to treatment outcome. In addition, cell culture studies were performed, evaluating the effect of BRAF/NRAS status on dacarbazine sensitivity.

RESULTS: We identified two BRAF splice variants lacking key parts of the catalytic domain (exon 12-15 and exon 14-15, respectively). Splice expression and BRAF V600E mutation status were mutually exclusive (p=0.006). All patients obtaining an objective response to chemotherapy expressed splice variants (p=0.014 and p=0.043 at 6 and 12 weeks, respectively). siRNA-mediated BRAF knockdown increased dacarbazine sensitivity in a melanoma cell line wt for BRAF (SBcl2) but not in SK-MEL 28, harbouring BRAF V600E. No correlation between BRAF or NRAS mutation status and treatment response was recorded.

CONCLUSION: Our findings suggest that BRAF plays a key role conferring resistance to dacarbazine treatment in metastatic melanomas and the del 12-15 or del 14-15 splice variants to have BRAF antagonizing effects.

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 2567.
Abstract 2573: Molecular mechanism of cisplatin-based chemoresistance with human head and neck cancer: A translational clinical study
Authors: Yamashita Taku , Miyamoto Shunsuke , Hwang Wei-Ting , O'Malley Bert W. , Li Daqing
Published: February 2011

(Background) Cisplatin is a commonly used chemotherapy agent for many kinds of cancers including head and neck cancer. Chemoresistance to cisplatin significantly contributes to treatment failure in clinical practice. The MRE11/RAD50/NBS1 (MRN) complex is well known a major DNA repair system. Our previous in vitro study shows that enhanced DNA repairing by MRN is a critical molecular mechanism for cisplatin-based chemoresistance. The present study further investigates if the finding of enhanced DNA repairing is responsible for cisplatin-based chemoresistance with human head and neck squamous cell carcinoma (HNSCC) in an animal model and, more important, if this finding correlates to the clinical study with patients who suffer from HNSCC.

(Methods) An animal model with human HNSCC was used for this study. Tumor volume changes were measured before and after cisplatin treatment. Immunohistochemistry studies were carried out for detecting the expression levels of MRN and apoptosis. The chemoresistant profile of the tumor model was established depending on these studies. The patients with HNSCC who initially received cisplatin monotherapy along with the patients without having cisplatin treatment were investigated. Immunohistochemistry studies were carried out for detecting the expression levels of MRN and apoptosis. These results were carefully analyzed by using a linear regression model with random effects to compare with established animal tumor model and in vitro findings.

(Results) Our animal model with human HNSCC demonstrated that chemoresistance to cisplatin is associated with increased expression levels of MRN after cisplatin treatment (p<0.0001(MRE11), p<0.0001(RAD50), p<0.0001(NBS1)). In addition, the resistant tumors showed decreased levels of apoptosis after cisplatin treatment (p<0.0001). Our clinical study confirms these findings as shown that patients with HNSCC had increased expression of MRN when developing cisplatin chemoresistnace.

(Conclusion) It is strongly suggested that increased expression levels of MRN proteins after cisplatin treatment are associated with a resistant phenotype in the human HNSCC. Our study presents important findings that will facilitate a better understanding of the molecular mechanisms underlying tumor cell resistance to chemotherapy.

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 2573.
Abstract 2594: Reovirus replication in ovarian and peritoneal tumors after intravenous administration
Authors: Phelps Mitch A. , Cohn David E. , O'Malley David M. , Wei Lai , Wilkins Deidre , Campbell Angela , Schaaf Larry J. , Coffey Matthew C. , Villalona-Calero Miguel A. , Grever Michael R. , Nuovo Gerard J. , Zwiebel James A.
Published: February 2011

Background: Reovirus Serotype 3 - Dearing Strain is an oncolytic virus under clinical evaluation for treatment of solid tumor malignancies. We initiated a phase 1/2 study with reovirus in patients with ovarian, primary peritoneal and fallopian tube carcinoma. The objectives of the correlative studies in this trial include the verification of reovirus penetration and replication in ovarian and peritoneal tumors after IV and IP reovirus administration and to evaluate tumor RAS pathway activation status as a marker for reovirus activity in this patient population.

Methods: Percutaneous tumor biopsies and ascites fluid were collected at various times during therapy from patients receiving reovirus via both IV and IP routes. Biopsied tissue and cell preparations from ascites were evaluated for reovirus capsid particles via electron microscopy (EM) and viral protein via immunohistochemistry (IHC). The presence of viral RNA and transcribed DNA are evaluated via reverse-transcriptase in situ PCR (RTISPCR). IHC for phosphorylated ERK (pERK) is applied to determine RAS pathway activation status.

Results: To date, 5 patients have been accrued and treated on trial. Preliminary evaluation with IHC indicates the presence of viral protein in the biopsied ovarian and peritoneal tissue samples from a subset of these patients after IV administration of reovirus. Completion of EM, RTISPCR, and pERK IHC on biopsied tissues and ascites samples is in progress.

Conclusions: Results to date display evidence of viral replication in peritoneal and ovarian cancer cells after IV administration of reovirus. This data represents the first observation of reovirus penetration into the peritoneal space after systemic drug administration. Completion of correlative analyses will further characterize the ability of reovirus to penetrate and replicate within the IP space and ascites after IV and IP administration. Furthermore, the selective viral infection and replication within RAS-pathway-activated tumor cells will be assessed. Results of all correlatives will be presented at the meeting. This work is supported by the NCI, grant U01CA076576 and contract NO1CM62207.

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 2594.
Abstract 2605: CE-TOFMS metabolome analysis for pharmacological responses to CPT-11 in colorectal cancer cells with different chemosensitivities
Authors: Nishimuta Akito , Suzuki Tetsuya , Arita Eri , Sugimoto Masahiro , Soga Tomoyoshi , Tanigawara Yusuke
Published: February 2011

Purpose: The topoisomerase I inhibitor irinotecan (CPT-11) is widely used for the treatment of metastatic colorectal cancer. However, mechanism to determine pharmacological response to CPT-11 is still unclear. We reported the application of a novel metabolomic analysis using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS) on the study of metabolic responses to anticancer agents (AACR #2563 and #4785, 2008 and #3764, 2009). The purpose of this study is to compare the metabolomic responses to SN-38, the active metabolite of CPT-11, between high-sensitive and low-sensitive colorectal cancer cells, for finding new biomarkers that can predict CPT-11 response.

Experimental Procedures: To elucidate metabolomic responses to SN-38, human colorectal carcinoma, HCT116 and HT29 cells were exposed to 50 nM SN-38. At time 0, 3, 8, and 24 h after SN-38 exposure, all intracellular metabolites were extracted by liquid-liquid extraction and simultaneously analyzed by CE-TOFMS. To find out low molecular weight compounds related to the sensitivity to SN-38, the metabolomic profiles were compared across the 8 colorectal cancer cell lines and sensitivity to SN-38 was investigated.

Results: The HCT116 cells were more sensitive to SN-38 than HT29. The intracellular metabolites of key pathways for cellular activity were quantitatively analyzed by CE-TOFMS, which included glycolysis, tricarboxylic acid (TCA) cycles, urea cycles, amino acid metabolism and pyrimidine metabolism. The intracellular levels of endogenous deoxyribonucleotides (dTTP and dCTP) increased after SN-38 exposure in both cells. In contrast, ribonucleotides such as CTP and UTP of RNA synthesis pathway were not affected by SN-38 exposure in either cell. These results imply the change in deoxyribonucleotide pool due to the disruption of DNA synthesis by topoisomerase I inhibition, the main anti-cancer mechanism of SN-38. It was also found that lactate and glycerol-3-phosphate were accumulated to a greater extent in HCT116 than in HT29. Furthermore, correlation analysis using 8 colorectal cancer cell lines revealed that the intracellular level of γ-aminobutyric acid (GABA) strongly correlated with cellular sensitivity to SN-38.

Conclusions: The present CE-TOFMS metabolome analysis created a new finding on metabolomic mechanisms for response to SN-38 by colorectal cancer cells with different chemosensitivities. GABA was found to be a potential low molecular weight biomarker that may be used for predicting CPT-11 response.

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 2605.
Abstract 2608: An intronic polymorphism determines TGF-alpha expression and is associated with cellular resistance to erlotinib
Authors: Wanqing Liu , Ratain Mark J.
Published: February 2011

An intronic polymorphism determines TGF-alpha expression and is associated with cellular resistance to erlotinib

Wanqing Liu, Mark J Ratain

Section of Hematology/Oncology, Department of Medicine, The University of Chicago, Chicago, IL 60637, USA.

Background

Responsiveness to EGFR-targeting agents has been demonstrated in lung cancer patients bearing EGFR mutations. However, the detailed mechanism underlying both sensitivity and resistance to these agents has not been completely revealed. Expression of transforming growth factor alpha (TGFA), one of the major EGFR ligands, appears to affect the inhibition potency of EGFR inhibitors. The aim of this study is to identify the genetic variants that may affect TGFA expression and confer susceptibility to response to EGFR-targeting agents.

Methods

Genome-wide genotyping of 100K single nucleotide polymorphisms (SNPs) had been previously performed in the NCI-60 cancer cell panel. SNPs in the TGFA gene region were selected to perform correlations with TGFA mRNA expression measured by real-time PCR. Confirmation of the SNP genotypes was performed by both sequencing and restriction enzyme digestion. The SNP genotype was correlated with cytotoxicity data of erlotinib and other EGFR inhibitors.

Results

A total of 10 SNPs in the TGFA gene were genotyped on the 100K SNP Chip. The genotypes were confirmed. A SNP (rs3821262 C/T) in the second intron of TGFA was significantly associated with decreased TGFA gene expression and cellular resistance to both erlotinib (p=0.01) and other 11 EGFR inhibitors (p=0.05) tested in the NCI-60.

Conclusion

The SNP rs3821262 C/T may contribute to cellular resistance to EGFR inhibitors by modulating the gene expression. However, since this is a hypothesis-generating study and the association was not adjusted for multiple testing, the finding must be validated further in both preclinical and clinical settings, in particular combined with mechanistic studies.

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 2608.
Abstract 2609: Nelfinavir induces liposarcoma apoptosis through inhibition of regulated intramembrane proteolysis of sterol regulatory element binding protein-1 and activating transcription factor 6
Authors: Guan Min , Jiang Chunling , Fousek Kristen N. , Guo Song , Chow Warren
Published: February 2011

We previously reported that nelfinavir induces a G1 cell cycle block and apoptosis selectively in liposarcoma cell lines as a consequence of up-regulation of SREBP-1 expression. We demonstrate now that nelfinavir-induced up-regulation of SREBP-1 and ATF6 is a direct result of inhibition of site-2 protease, which together with site-1 protease mediates regulated intramembrane proteolysis from their precursor state to their transcriptionally-active forms. The resulting endoplasmic reticulum stress and concurrent inhibition of the unfolded protein response induces caspase activation. Time-lapse, confocal microscopy studies demonstrate that nelfinavir inhibits the normal translocation of transfected SREBP-1-EGFP and ATF6-EGFP fusion proteins from the cytoplasm to the nucleus. siRNA-mediated knockdown of site-1 and/or site-2 protease leads to inhibition of SREBP-1 intracellular trafficking to the nucleus and reduces liposarcoma cell proliferation. Treatment of LiSa-2 liposarcoma cells with 4-(2-aminoethyl)benzenesulfonyl fluoride, a serine protease inhibitor of S1P did not affect SREBP-1 processing. In contrast, 1, 10-phenanthroline, an S2P-specific inhibitor, reproduces the molecular and biological phenotype observed in nelfinavir-treated cells, which confirms S2P is a target of nelfinavir. In vivo evaluation of nelfinavir in a murine liposarcoma xenograft model shows inhibition of tumor growth in the absence of significant toxicity. These results provide new insight into the mechanism of nelfinavir-mediated induction of ER stress and cell death in liposarcomas and are the first report to therapeutically target S2P against 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 2609.