bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4219: Lysosomal membrane permeabilization, a novel anticancer mechanism induced by pterostilbene
Authors: Mena Salvador , Rodriguez Maria , Asensi Miguel , Estrela Jose M. , Ortega Angel
Published: July 2012

Pterostilbene (Pter) (3,5-dimethoxy-4′-hydroxystilbene), a natural dimethylated analog of resveratrol, is a phytoalexin abundant in plants and fruits with a number of potential benefits for human health. Darakchasava, an Indian herbal preparation of Vitis Vinifera, contains Pter and is prescribed as a cardiotonic in ayurvedic and traditional medicine. Furthermore, some observations indicate that Pter can be beneficial in the prevention and treatment of different diseases such as diabetes, dyslipidemia, or cancer.

Pter shows higher bioavailability than resveratrol. The substitution of two OH groups (positions 3 and 5) by methyl groups increases the stabilityof the molecule and its resistance to hepatic glucuronidation and sulfonation. Nevertheless, despite these pharmacokinetic advantages, the anticancer potential of Pter is still undefined.

Lysosomal membrane permeabilization (LMP) has been postulated as a mechanism leading to cell death. In an initial step LMP causes cytosol acidification and release of cathepsins and other hydrolases from the lysosomal lumen. Ectopic presence of proteases causes the hydrolysis of vital proteins and the activation of other cell death-related proteases such as caspases. Thus linking LMP and cell death type I. In addition, LMP may also induce caspase-independent cell death mechanisms. In fact morphology of cells affected by LMP may be confused with a subapoptotic or necrotis appearance. The aim of our present study was to investigate the mechanisms of cell death activated by Pter in different tumor cells. For this purpose we used a range of Pter concentration between 10uM (below the IC50) and 100uM (an unachievable in vivo concentration). Acridin orange staining was determined by flow cytometry and fluorescence microscopy. Total and cytosolic cathepsin extractions were performed with different digitonin concentration and activity was measured by spectrofluorimetry. Cytosolic pH was evaluated by staining cultured cells with the fluorochrome SNARF. Apoptosis induction was checked through caspase 3 and 7 measurements.

The effects observed in the presence of Pter, under in vitro conditions, were concentration and time dependent. An increase in LMP induced an acidification of cytosolic pH, an increase in cathepsin activity and, only at long time exposure, apoptosis stimulation. Our results demonstrate that tumor cell death induction elicited by Pter is preferentially mediated through LMP.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4219. doi:10.1158/1538-7445.AM2011-4219
Abstract 4221: Inhibition of hedgehog signaling by extracts of sutherlandia
Authors: Lu Yuan , Lin Hui , Drenkhahn Sara K. , Starkey Nicholas J.E. , Jackson Glenn A. , Folk William , Fritsche Kevin L. , Lubahn Dennis B.
Published: July 2012

Many botanical compounds that have been proposed to prevent cancer may potentially work via inhibition of the hedgehog-signaling pathway. Here we investigated the potential of Sutherlandia frutescens (also called “cancer bush” in South Africa) to prevent and/or treat prostate cancer. We hypothesize that the anti-cancer effects of Sutherlandia are due to its inhibition of hedgehog-signaling pathway activity. To determine hedgehog pathway inhibitory activity, we treated Shh light II cells with multiple doses of a methanol extraction of Sutherlandia and measured Gli1 reporter activities.

Results: We found that a methanol extract of Sutherlandia was able to inhibit hedgehog pathway activity in a dose-dependent manner as monitored by Gli reporter assay (IC50=1:4000). Moreover, the Sutherlandia extract can inhibit the growth of human prostate cancer cells PC3 and LNCaP with IC50 of 1:400 and 1:1500 fold dilutions, respectively. At these same extract dilutions normal prostate cancer cell growth was not inhibited. Our data indicate that Sutherlandia contains potent anti-cancer botanicals that have hedgehog inhibitory activity.

Conclusion: Our results suggest that this plant offers a potentially cheap and effective alternative for hedgehog-driven cancer therapies. Additionally, Sutherlandia may yield novel targets that potentially could lead to a second generation hedgehog inhibitor, as resistance has been found to the first generation drugs currently in clinical trials.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4221. doi:10.1158/1538-7445.AM2011-4221
Abstract 4225: Thymoquinone induces cell death in glioblasotma cell through production of reactive oxygen species and induction of DNA damage
Authors: Arafa El-Shaimaa , Racoma Ira , Wani Gulzar , Wani Altaf
Published: July 2012

Glioblastoma is the most aggressive and common type of brain tumors in humans. Surgical resection followed by radiation and chemotherapy is the current standard treatment; however, due to up-regulation of several survival pathways, tumor recurrence is quite common with a median survival of 12 to 15 months. Therefore, there is an urge for more effective therapeutic strategies for the treatment of glioblastoma. Over years the use of natural compounds as anticancer agents acquired great importance. Thymoquinone (TQ) is the bioactive component of the volatile oil extracted from the black seed, Nigella sativa. TQ has shown anti-oxidant, anti-inflammatory, and anti-neoplastic actions in in-vitro and in-vivo models with a selective cytotoxicity for human cancer cells compared to several normal cells. Here, we report that TQ greatly inhibited cell proliferation of radioresistant glioma cells (U87 and T98) in a concentration-dependent manner. Moreover, TQ mediated reactive oxygen species-induced DNA breaks in glioma cells, as detected by the appearance of post-DNA damage sensitive marker γH2AX. Our results also showed that TQ up-regulated p53 in U87 cells while down-regulated phospho-Akt in T98 cells in a dose-dependent manner. Current investigations are ongoing to use TQ in combination with ionizing radiation as a promising modality to overcome resistance, thereby reducing ionizing radiation doses used for glioma treatment. This work was supported by NIH grants CA93413, ES2388 and ES12991 to A. A. W.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4225. doi:10.1158/1538-7445.AM2011-4225
Abstract 4250: A novel thrombospondin-1 mimetic peptide, ABT-898, decreases angiogenesis in a mouse model of glioblastoma multiforme
Authors: Yamada Ryosuke , Nakano Ichiro
Published: July 2012

Prognostic outlook for patients with glioblastoma multiforme (GBM) still appears bleak in spite of medical advances in treatment of other malignant tumors in the past decades. With optimal therapy, of surgical resection, radiation therapy, and chemotherapy, a median survival merely approximates to 12 months. However, a recent dogma that the microvascular niche of such tumors plays a critical role in their maintenance has led to development of “anti-angiogenenic” therapies that target it. To evaluate the apoptotic effect of ABT-898, a TSP-mimetic, anti-angiogenic peptide on endothelial cells, we used human umbilical vein endothelial cells (HUVEC) to model human brain microvasculature. Furthermore, to then assess its clinical applicability, an orthotopic xenograft mouse model was established using human-derived GBM cells lines, and its therapeutic effect evaluated in vivo. We found that ABT-898 induced apoptosis on HUVEC as evidenced by increased fraction of annexin-V positive cells on flow cytometry. Systemic treatment of BTSC-derived immunodeficient mouse tumors with ABT-898, either alone or in combination with temozolomide, significantly reduced tumor size at day 14 post-injection of GBM cells, compared with that of a vehicle, which was used as a control. In addition, treatment with ABT-898 allowed increase in median survival in the xenograft models compared to the control mice. These observations suggest that ABT-898 is a plausible candidate for use in future treatment of GBM.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4250. doi:10.1158/1538-7445.AM2011-4250
Abstract 4260: Sunitinib treatment induces Notch expression and activation in renal carcinoma cell lines
Authors: Ziouti Fani , Touplikioti Panagiota , Kontovini Maria-Ekaterini , Vadeka Christina , Kontovinis Loukas , Papazisis Konstantinos
Published: July 2012

Introduction. Notch signaling is an evolutionary conserved pathway that plays a central role in stem cell biology, tumor formation, angiogenesis and cell fate decisions. Currently there is not any clear view as to whether Notch plays an oncogenic role in renal cell cancer (RCC). Notch-1 is activated after cytotoxic stimuli and may induce mTor pathway activation, leading to increased cell survival.

We have investigated the role of Notch expression and activation in RCC cell lines in response to sunitinib treatment and the role of Notch in the activation of the mTor pathway. We elaborated three cell lines with a different VHL and PTEN mutation status: Caki-1 cells (VHL-wt, PTEN-wt), Caki-2 (VHL-mut, PTEN-wt) and 786-O (VHL-mut, PTEN-mut).

Results: When Caki-1 and Caki-2 cells were exposed to sunitinib there was a marked upregulation in the expression of the Notch-1 mRNA and the target transcription factors RUNX1, RBPJ and HES1. The PI3K – mTor – S6K pathway was aslo upregulated. This upregulation was reversed when cells were pre-treated with a gamma-secretase inhibitor. However, both gamma secretase inhibitors and everolimus (an m-Tor inhibitor) had an antagonistic effect when combined with sunitinib (Combination Index >> 1.0). On the other hand, PTEN-mutant 786-O cells have a dysregulated mTor pathway and are more sensitive to inhinition of the m-Tor with everolimus. Sunitinib exposure did not result in further increase of the PI3K-mTor-S6K activation. Interestingly, combination of sunitinib with everolimus was synergistic in 786-O.

Conclusions: VHL-wt and VHL-mutant RCC cells treated with sunitinib activate the Notch pathway. There are reports that Notch plays a tumor suppressor role in RCC and our results are on concert with that. Furthermore, Notch seems to regulate m-Tor pathway activation in cells with a wild-type PTEN.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4260. doi:10.1158/1538-7445.AM2011-4260
Abstract 4270: Targeting cancer cells resistant to hypoxia and nutrient starvation double-deprivation stress as a novel cancer therapy
Authors: Osawa Tsuyoshi , Muramatsu Masashi , Tsuchida Rika , Wang Feng , Yuasa Yasuhito , Shibuya Masabumi
Published: July 2012

Anti-angiogenesis is an effective cancer treatment, however, tumor progression and refractoriness to anti-angiogenesis are observed in both clinical and preclinical cancer models. Stress under hypoxia and nutrition deprivation might play an important role in the tumor aggressiveness and refractoriness. Here we show that a long term hypoxia and nutrient starvation double-deprivation stress (DDS) stimulates migration, invasion and anchorage independent growth of tumor cells in vitro and an increased tumor progression and angiogenesis in vivo associated with an increase in blood vessels and infiltration of CD11b+ cells. We also observed up-regulation of DDS-responsive genes including VEGF-A, FGF18, and JHDM2A. We found that histone demethylase JHDM2A is commonly up-regulated under DDS in human and mouse cancer cell lines in vitro and in vivo at the phase of pre-angiogenic switch and refractory phase of anti-angiogenic treatments. Specific inhibition of JHDM2A by siRNAs significantly suppressed tumor growth in vivo. Importantly, suppression of JHDM2A demonstrated synergetic effects in treatment with anti-VEGF neutralizing antibody (bevacizumab) or small molecular inhibitor for VEGF receptors (sunitinib), and improved an efficacy of anti-angiogenesis. Collectively, our results indicate that minor population of cancer cells resistant to both hypoxia and low nutrition acquires tumor aggressiveness through specific mechanisms upon the DDS responses. Our observations suggest that the targeting of cancer cells resistant to hypoxia and nutrient starvation by inhibition of DDS-responsible genes such as JHDM2A can be utilized for a novel treatment to overcome the refractoriness to anti-angiogenic therapy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4270. doi:10.1158/1538-7445.AM2011-4270
Abstract 4278: Spheroid growth in 3D alginate matrices
Authors: Dornish John Michael , Andersen Therese , Heier-Baardson Helene , Markussen Christine , Ward Carol , Mullen Peter , Langdon Simon , Kunkler Ian , Harrison David
Published: July 2012

The use of 3-dimensional (3D) matrices for cell culture is gaining popularity as a substitute for traditional 2D cell culture as it can approximate cell architecture and cell-cell contact found in tissues, organs and tumors. NovaMatrix-3D™ is an alginate-based cell culture system comprising an alginate foam matrix and an alginate immobilizing solution. In principle, cells are first suspended in a sodium alginate solution then the cell suspension is applied to the alginate foam. In situ gelation occurs when calcium ions are donated from the foam cross-linking the added alginate, effectively entrapping the cells within the pores throughout the foam. To show the utility of the NovaMatrix-3D™ culture system, various cell lines were first cultured in 2D then prepared as a suspension in 0.5 – 1% sodium alginate in cell culture medium. An aliquot of each cell suspension was added to γ-sterilized NovaMatrix-3DTM foams (12- or 24-well plate size). NHIK 3025 (cervix carcinoma), MCF7 (breast adenocarcinoma), ZR-75-1 (breast ductal carcinoma) and NIH:OVCAR-3 (ovarian adenocarcinoma) were used and single cells were immobilized in the alginate foams. Cell localization within the foam was visualized using confocal microscopy to identify fluorescently labeled (CellTrace™ CFSE) cells. Cell proliferation was measured by counting cells after de-gelling the foam using sodium citrate. In each case, cells were immobilized with or without RDG-coupled alginate to investigate the importance of the presence of cell attachment peptides within the alginate. For these cell lines, cell growth and multicellular spheroid formation was independent of the presence of RGD. Spheroids could also be removed intact by de-gelling the alginate matrix and further processed for histological staining. One example shows the selective staining of apoptotic cells within the spheroid. Use of alginate foams with concomitant in situ immobilization of cells results in a 3D cell culture model with the potential to approximate cell proliferation and architecture within tissues or tumors. The technology enables biomimetic approaches by varying e.g. matrix elasticity, gelling ions, attachment peptides and foam degradation making NovaMatrix-3D™ a versatile cell culture system. Portions of this work have been funded with the support from METOXIA project no.222741 under the 7th Research Framework Programme of the European Union.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4278. doi:10.1158/1538-7445.AM2011-4278
Abstract 4296: Activated ALK accelerates the onset of neuroblastoma in MYCN-transgenic zebrafish
Authors: Zhu Shizhen , Lee Jeong-Soo , Shin Jimann , Perez-Atayde Antonio R. , Kutok Jeffery L. , Rodig Scott J. , Neuberg Donna S. , Guo Feng , Helman Daniel , Feng Hui , Stewart Rodney A. , Wang Wenchao , George Rani E. , Kanki John P. , Look A. Thomas
Published: July 2012

Neuroblastoma is the most common extracranial solid tumor in children and originates during development of the peripheral sympathetic nervous system. Advanced-stage disease and a poor outcome are associated with amplification of the MYCN oncogene, often in combination with mutational activation or amplification of another oncogene ALK (anaplastic lymphoma kinase), suggesting that they cooperate in neuroblastoma pathogenesis. To investigate this possibility, we generated a transgenic zebrafish model of neuroblastoma in which human MYCN is expressed under the control of the dopamine-beta-hydroxylase promoter, and show that the resultant tumors recapitulate childhood neuroblastomas histologically, immunohistochemically, and ultrastructurally. Surprisingly, the tumors arise from a subpopulation of neuroblasts that migrate into the adrenal analogue (interrenal gland) in the zebrafish after organogenesis is complete. Coexpression of activated ALK with MYCN markedly increased the frequency of neuroblastoma and accelerated the time of onset, providing conclusive evidence for synergistic interplay between these two oncogenes in neuroblastoma pathogenesis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4296. doi:10.1158/1538-7445.AM2011-4296
Abstract 4297: Morphology of MCF-7 colonies formed in agarose cell culture
Authors: Aulthouse Amy L. , Nino Elizabeth Di , Kinder David H.
Published: July 2012

Previously, we demonstrated that the MCF-7 cell line grows in a permissive 3-D agarose culture (Kinder and Aulthouse, Cancer Letters., 2004, 205(1), 49-53). Single cells form colonies over time; thus, mitotic activity can be monitored in response to various agents. We studied the morphology of the colonies that formed following growth from single cells at 3 time points: 1.5, 2, 2.5 weeks. The MCF-7 cells grew into clusters (solid) and hollow tube-like structures. These structures were confirmed by H&E stained serial sections of paraffin embedded cultures. H&E staining also revealed abundant mitotic figures. Because calcium foci are common in breast cancers, we also evaluated the cultures for calcium expression using the von Kossa reaction. Placenta was used as a control. At all time points, calcium was detected, but no particular pattern was noted in the calcium distribution between the clusters and the hollow tube-like structures. With morphology and calcium expression described, we further examined cell growth inhibition and cytotoxicity of estradiol and tamoxifen on the MCF-7 cells. Added estradiol enhanced the growth of the cells, while tamoxifen was cytostatic at concentrations below 1.3 × 10-4 nM. At high tamoxifen concentrations (>2 × 10-4 nM) cytotoxicity was noted. This was likely due to sigma-2 receptor binding rather than estrogen receptor blocking. Combination of estradiol with tamoxifen had a more pronounced effect on the cytotoxicity caused by tamoxifen. These morphological and mechanistic findings support the use of this model for studying the MCF-7 cell line in agarose culture.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4297. doi:10.1158/1538-7445.AM2011-4297
Abstract 4310: Gene expression analysis of axial and appendicular osteosarcoma in the canine model
Authors: Dervisis Nikolaos , Klahn Shawna L.
Published: July 2012

Spontaneously occurring canine osteosarcoma (OSA) is considered an excellent model of the human disease. Canine axial and appendicular osteosarcoma appear histopathologically identical. The clinical course of the two forms, however, seems to diverge. Following surgery, the axial form may be associated with a lower metastatic rate and longer survival than the appendicular form. Identifying gene expression profiles associated with decreased metastatic potential may be crucial for the development of targeted therapies for the human disease. We hypothesized that there are distinct gene expression patterns for canine axial and appendicular OSA.

Six axial osteosarcoma tumor samples and six age, breed, and weight matched appendicular osteosarcoma clinical tumor samples were selected from the Center for Comparative Oncology, Michigan State University tissue bank. RNA extraction, amplification and labeling were performed using standard techniques. Raw gene expression values were obtained using the Affymetrix GeneChip Canine Genome 2.0 Array. Background correction, normalization, PM correction, summarization, and expression analysis were performed using the R software with the affy/limma/sva packages. Gene set enrichment analysis was performed using GSEA software.

All RNA samples passed quality control and were included in the data set. The expression analysis took into account RNA degradation, which was similar in all tumor samples. A total of 184 genes were differentially expressed by >16-fold, between the two groups. Surrogate variable analysis did not result in any statistically significant differences in gene expression. Gene set enrichment analysis resulted in 9 gene sets enriched in axial, and 4 gene sets enriched in appendicular osteosarcoma samples at FDR < 25%. Gene ranking was similar between the two methods of analysis, with MMP1, ATP6AP1, SLC6A8, and dysadherin expression increased in axial tumor samples, while ICAM2 expression increased in appendicular samples.

Expression analysis indicates no statistically significant differences in gene expression between the axial and appendicular OSA. Gene set enrichment analysis indicates potential differences in gene set enrichment between the two types of osteosarcoma. Further quantitative RT-PCR analysis is required to confirm our results. These data will guide hypothesis-driven translational studies, which may have direct clinical implications on the treatment of osteosarcoma.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4310. doi:10.1158/1538-7445.AM2011-4310
Abstract 4318: A constitutively active NF-κB2 mutant drives plasma cell tumor development in mice
Authors: Yang Liqun , McCarthy Brian , Ding Jane , Ding Han-Fei
Published: July 2012

NF-κB2 is a member of the NF-κB family of transcription factors. Genetic mutations that lead to the constitutive action of NF-κB2 signaling occur in approximately 20% human multiple myeloma, a common malignant tumor of plasma cells. Some of the mutations occur in the NF-κB2 gene, leading to the generation of C-terminally truncated NF-κB2 mutants that are constitutively active transcription factors. We investigated the pathogenic role of NF-κB2 mutation in the development of plasma cell tumors using a transgenic mouse line with targeted expression of a tumor-derived NF-κB2 mutant, p80HT, in lymphocytes. The mutant mice developed predominantly B cell-lineage lymphomas, with half of them being plasma cell tumors and producing monoclonal Ig. Some of the plasma cell tumors display key features of human multiple myeloma, including a marked increase in bone marrow plasma cell content, osteolytic bone lesions and/or diffuse osteoporosis. Gene expression profiling of splenic B cells from monoclonal Ig-producing p80HT transgenic mice and their wild-type littermates revealed 740 genes that were differentially expressed (575 upregulated and 165 downregulated) in p80HT B cells versus wild-type B cells. Among the genes selectively upregulated in p80HT B cells are those known to be relevant to human multiple myeloma pathogenesis, including cyclin D1, cyclin D2, CD27, CD80, IL10, and survivin. The upregulation of cyclin D1, IL10 and survivin was confirmed by immunoblotting and/or real-time PCR. Chromatin immunoprecipitation and quantitative PCR assays demonstrated direct interaction of p80HT with the surviving gene promoter in vivo. These findings provide direct evidence for a causal role of NF-κB2 mutation in the pathogenesis of plasma cell tumors in mice and suggest a molecular mechanism underlying the tumorigenic activity of NF-κB2 mutation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4318. doi:10.1158/1538-7445.AM2011-4318
Abstract LB-163: Immunotherapy utilizing MUC-1-messangerRNA encoding DC and MUC-1 specific CTL in combination with Gemcitabine targeting unresectable or recurrent pancreatic cancer
Authors: Yoshimura Kiyoshi , Maeda Yoshinari , Shindo Yoshitaro , Watanabe Yusaku , Yoshino Shigefumi , Hazama Shoichi , Oka Masaaki
Published: July 2012

Background; Mucin1 (MUC1) is known as a mucin core protein expressing in inv asive ductal carcinomas of the pancreas. MUC1-specific cytotoxic T lymphocyt es (CTLs) recognize MUC1 moleculesin a HLA-unrestricted manner. We have prev iously reported that the adoptive immunotherapy (AIT) utilizing MUC1-CTLs stimulated with a MUC1-expressing human pancreatic cancer cell line, named Y PK-1, was useful and safe. We also reported the development of an AIT of MUC 1-CTLs in combination with dendritic cells (DCs) pulsed with MUC1-peptide as well.

Here we report that the therapy using DC encoding MUC1-mRNA and MUC1-CTLs targeting unresectable or recurrent pancreatic cancer is safe and effective.

Purpose; We assessed whether this immunotherapy is safe and effective.

Objective and Methods; Twenty patients with unresectable or recurrent pancreatic cancer had the therapy what we planned, one week later, peripheral blood mononuclear cells (PBMC) were harvested by leukapheresis. The PBMCs were separated and induced to be DCs and CTLs. Matured DCs received MUC-1-mRNA by electroporation, then administrated via subcutaneously. CTLs were co-cultured with YPK-1 pancreatic cancer cell line expressing MUC-1 under the stimulation with IL-2, then administrated via intravenously.

Results; Male/Female ratio was 1 to 1 (10/10), Mean age was 60.6 y.o. (37–78), Mean times of administration of cells was 3.35 (1.5–27.9), Mean number of c ells that were administrated was 46 million (DCs) and 1800 million (CTLs), Median observation period was 16.2 months (1.5–27.9). One-year survival rate was 39% (median survival time, 6.9 months). When we focus on the patients with unresectable cancer, their one-year survival rate was 54% (median surviva 1 time, 18.4 months). In addition, patients received DCs of more than 10 million had 58% of one-year survival rate, whereas no patient received less than 10 million's DCs did survive in one year (median survival time, 5.8 months) (p=0.048, Logrank). All of patients received the therapy as outpatients and showed no side effect.

Conclusion; First of all, this therapy is quit safe. Second, patients received DCs of more than 10 million had high one-year survival rate suggesting high volume DCs in this combination therapy had high potential.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-163. doi:10.1158/1538-7445.AM2011-LB-163
Abstract LB-165: Low TCR diversity (divpenia) is a new prognosis factor of overall survival in metastatic breast cancer
Authors: Manuel Manuarii , Tredan Olivier , Bachelot Thomas , Rabeony Tioka , Chabaud Sylvie , Mouret Jean-François , Grives Audrey , Perez Solène , Puisieux Alain , Blay Jean-Yves , Caux Christophe , Pasqual Nicolas , Caux Christine Menetrier
Published: July 2012

Rationale

Lymphopenia (<1000 Lymphocytes/μl) or CD4+ T cell lymphopenia (<450/μl), detected before initiation of chemotherapy are predictive factors for toxicity and death in metastatic solid tumors (Borg et al 2004; Ray Coquard et al 2009).

The goal of the present study was to further identify the characteristics of the T cells in these lymphopenic patients. TCR diversity was investigated and tested as predictive factor for overall survival (OS).

Patients and methods

The ImmunTraCkeR® assay (ImmunID, Grenoble France), which analyzes, through semi quantitative multiplex PCR, the V-D-J combinatorial diversity of TCR-beta chain (TRB), was used to investigate diversity of T cell repertoire on cryopreserved blood samples from a prospective cohort of untreated metastatic breast cancer patients (SEMTOF study)(n=66). Univariate and multivariate analysis of prognostic factors for OS were investigated in this series as well as in a validation series.

Results

A severe T cell Divpenia (T cell diversity below 33%) (average diversity for healthy people is 70%) was associated with median survival of 9 months vs 24 months for the remaining patients (logrank p.value=0.0047). In a multivariate analysis, including haemoglobin level, polynuclear neutrophil count and liver metastasis, divpenia (defined at 30% threshold defined by ROC curve analysis) was identified as an independent prognostic factor. The NDL® score (Numeration Diversity Lymphocytes representation) that combines lymphocytes numeration with TRB diversity, demonstrated that lympho-divpenia (T cell diversity below 33% and lymphopenia at 1Giga/L threshold) was associated with a poor prognosis in term of patients survival compared to patients with either a good numeration or good diversity or both good numeration and diversity (p.value=0.0202). Prognostic value of NDL® score was then evaluated on a prospective validation cohort of 33 patients (logrank p.value=0.002).

Conclusion:

Divpenia, and a combined parameter NDL are predictive factors for survival in metastatic breast cancer patients. An open clinical study (LYMPHOS1) will investigate divpenia and NDL® score in a larger prospective cohort of metastatic breast patients.

Acknowledgments

M.MANUEL is a CIFRE recipient. This work (Proof of Concept “LYMPHOS1”) was financially supported by CLARA (LYON, France)

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-165. doi:10.1158/1538-7445.AM2011-LB-165
Abstract LB-169: Dissecting signaling pathways regulating translation in human glioblastoma
Authors: Grzmil Michal , Hemmings Brian
Published: July 2012

Glioblastoma multiform (GBM) is the most common and aggressive brain tumor entity, with a median survival of approximately one year. In the last decade, genetic profiling of brain tumors has improved our understanding of gliomagenesis and proposed many targeted therapies based on molecular interference with deregulated signaling networks. In a search for novel molecular targets, our kinome-focused microarray analysis identified overexpressed MAP kinase-interacting kinase 1 (MNK1) in primary GBM and its elevated protein level was confirmed in primary GBMs and in glioma cell lines. MNKs can bind to translation initiation factor eIF4G and phosphorylate the cap-binding protein eIF4E at Ser209 that is required for its oncogenic activity. Targeting MNK1 activity in GBM cells suppressed eIF4E phosphorylation, reduced proliferation and colony formation whereas, concomitant treatment with Mnk inhibitor CGP57380 and mTOR inhibitor rapamycin resulted in an additive effect on growth inhibition and cell cycle arrest. Analysis of polysomal profiles revealed inhibition of translation in CGP57380 and rapamycin treated cells. Microarray analysis of total and polysomal RNA from MNK1-depleted GBM cells identified mRNAs involved in regulation of TGF-β pathway. The translation of SMAD2 mRNA as well as TGF-β-induced cell motility was regulated by MNK1-signaling. TGF-β pathways regulate growth of glioma-initiating cells that are involved in tumorigenesis, resistance to cancer therapies and relapse. Our most recent analysis identified high activity of MNK1 pathway in human GBM-derived spheres. Furthermore, inhibition of MNK1 activity significantly reduced formation and growth of cancer spheres. Our model of gliomasphere growth regulation via MNK1-signaling pathways together with clinical implications will be presented and discussed.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-169. doi:10.1158/1538-7445.AM2011-LB-169
Abstract LB-172: Role of post-translational modification of the RUNX2 transcription factor in osteosarcoma
Authors: Ito Tatsuo , Xinyang Zhao , Suehara Yoshiyuki , Kawai Akira , Nimer Stephen D. , Ladanyi Marc
Published: July 2012

Background-Several transcription factors or co-factors have recently been reported to be methylated at certain arginine residues, a post-translational modification mediated by two classes of protein arginine methyltransferases (PRMT). Moreover, some PRMTs have been shown to be key regulators of mesenchymal differentiation, and one of the PRMTs is involved in the epigenetic programming of early embryo development. RUNX2 is major transcription factor in osteoblast differentiation but little is known about its activity or function in osteosarcoma (OS). RUNX1, another member of RUNT family, is known to methylated at R206/R210, within the Runt domain, a region that has considerable homology with RUNX2. Here, we examined the possible role of PRMTs in OS in relation to the post-translational modification of RUNX2 and its possible impact on cell proliferation and differentiation.

Method-We generated arginine methylation-specific antibodies to RUNX2. The specificity of this antibody was confirmed by detecting immunoprecipitated RUNX2 from Saos2 cells by Western blot. We also confirmed the expression of RUNX2 and arginine methylated RUNX2 in Saos2 cell by immunofluorescence. We also examined the frequency of arginine methylation of RUNX2 in OS patient samples, compared to total RUNX2, by Western blot. Finally, we examined the influence of arginine methylation on the transcriptional activity of RUNX2 by luciferase assay using osteocalcin promoter plasmid.

Results-We found that PRMTs can methylate the arginine residue in the Runt domain of RUNX2. This arginine methylation of RUNX2 alters its transcriptional activity at the osteocalcin promoter.

Immunofluorescence with arginine methylation-specific antibody showed weak nuclear positivity in the SAOS-2 cell line. Patient tumor samples also show the expression of arginine methylated RUNX2, as well as total RUNX2.

Conclusion- Our results confirm that arginine methylation of RUNX2 affects its function as a transcription factor. Further study of the possible role of arginine methylation of RUNX2 in regulating its effect on osteosarcoma proliferation and differentiation could be of translational interest, given the therapeutic potential of PRMT inhibitors.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-172. doi:10.1158/1538-7445.AM2011-LB-172
Abstract LB-173: Genome-scale analysis of aberrant DNA methylation in colorectal cancer
Authors: Hinoue Toshinori , Weisenberger Daniel J. , Lange Christopher P.E. , Noushmehr Houtan , Byun Hyang-Min , van Dijk Cornelis M. , Pan Fei , Malik Simeen , Van Den Berg David J. , Shen Hui , Tollenaar Rob A.E.M. , Laird Peter W.
Published: July 2012

Colorectal cancer (CRC) is a heterogeneous disease in which unique subtypes are characterized by distinct genetic and epigenetic alterations. Here we performed comprehensive genome-scale DNA methylation profiling of 125 colorectal tumors and 29 adjacent normal tissues. We identified four DNA methylation-based subgroups of CRC using model-based cluster analyses. Each subtype shows characteristic genetic and clinical features, indicating that they represent biologically distinct subgroups. A CIMP-high (CIMP-H) subgroup includes all CIMP-positive tumors characterized by the MethyLight five-marker panel, and is strongly associated with MLH1 DNA hypermethylation and the BRAFV600E mutation. A CIMP-low subgroup is enriched for KRAS mutations and characterized by DNA hypermethylation of a subset of CIMP-associated markers rather than a unique group of CpG islands. Non-CIMP tumors are separated into two distinct clusters. One non-CIMP subgroup is distinguished by a significantly higher frequency of TP53 mutations and frequent occurrence in the distal colon, while the tumors that belong to the fourth group exhibit a low frequency of both cancer-specific DNA hypermethylation and gene mutations and are significantly enriched for rectal tumors. Furthermore, we identified 114 genes that were downregulated more than 2-fold in CIMP-H tumors together with promoter DNA hypermethylation. These represent approximately 7% of genes that acquired promoter DNA methylation in CIMP-H tumors. Intriguingly, 48/114 genes were also transcriptionally silent in non-CIMP subgroups, but this was not attributable to promoter DNA hypermethylation. Together, we identified four distinct DNA methylation subgroups of CRC and provided novel insight regarding the role of CIMP-specific DNA hypermethylation in gene silencing.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-173. doi:10.1158/1538-7445.AM2011-LB-173
Abstract LB-312: Methylated RASSF1a is the first specific DNA marker for minimal residual disease testing in neuroblastoma
Authors: Stutterheim Janine , Ichou Fatima Ait , den Ouden Emmy , Versteeg Rogier , Caron Huib N. , Tytgat Godelieve A.M. , Ellen van der Schoot C.
Published: July 2012

Purpose

PCR-based detection of minimal residual disease (MRD) in neuroblastoma is presently based on neuroblastoma-specific transcripts. However, the expression of these targets varies between patients and upon treatment, and only PHOX2B is truly specific. RASSF1a is methylated (RASSF1aM) in neuroblastoma and we investigated whether it can serve as a specific and stable DNA MRD marker.

Patients and methods

The RASSF1aM specific real-time quantitative (RQ)-PCR was tested on control bone marrow (BM) (n=50), on 71 neuroblastoma tumors and on 159 clinical BM samples at diagnosis and at follow up of 77 patients. Results were compared to a panel of RNA markers and correlated with prognosis.

Results

RASSF1aM was present in all stage 4 and 4s tumors (n=50) and in 86% stage 1–3 tumors (n=21). The level of methylation in stage 4 neuroblastoma was correlated with overall survival (p=0.02). RASSF1aM-PCR was highly specific (only one amplification in 50 control samples tested in triplicate) and had a similar sensitivity as the RNA-based PCRs as shown on clinical samples. Moreover, RASSF1aM enabled accurate quantification without need for the original tumor.

Conclusion

RASSF1aM is a novel, highly specific DNA marker for MRD detection in neuroblastoma, equal to PHOX2B in specificity and sensitivity, and better suitable for MRD quantification. We propose to include RASSF1aM in further prospective MRD studies in neuroblastoma alongside RNA MRD markers. In addition, this assay can be used for sensitive detection and quantification of circulating cells in all cancers with hypermethylated RASSF1a.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-312. doi:10.1158/1538-7445.AM2011-LB-312
Abstract LB-328: Highly sensitive detection of miRNA and mRNA from frozen, FFPE tissue and body fluid samples by expression microarray
Authors: Takizawa Satoko , Sudo Hiroko , Ueda Yoji , Kuroda Toshihiko , Ichikawa Makiko , Nomura Osamu , Akiyama Hideo
Published: July 2012

The RNA profiling from clinical samples readily obtainable in the routine care, such as blood or FFPE samples, is a promising method to find the unknown high-value diagnostic markers. However, as RNA is subjected to degradation even in properly-collected, and immediately frozen tissue, it is more difficult to obtain intact RNA from FFPE or body fluid samples for diagnostic analysis. “3D-Gene” is a highly sensitive gene expression microarray, featuring the unique pillar structure on the substrate and the beads agitation system during the hybridization reaction. Using “3D-Gene”, we achieved highly sensitive detection of mRNA or miRNA from diagnostically important clinical samples.

Total RNA was extracted from human serum, plasma and frozen or FFPE tissue samples, with the recommended protocol for each sample. For mRNA detection, total RNA was reverse-transcribed to cDNA and labeled with fluorescent dye directly or after the amplification. For miRNA detection, total RNA was labeled with fluorescent dye directly. These pretreated target nucleotides were hybridized to “3D-Gene” while the hybridized buffer containing target nucleotides was agitated by beads during hybridization. The hybridized microarrays were washed and scanned for image acquisition.

(1) With “3D-Gene”, we could detect the mRNA expression profile from FFPE samples with high reproducibility. We also showed high correlation of the expression profiles between FFPE and frozen tissue samples. With 500 ng total RNA obtained from frozen as well as FFPE tissue samples, miRNA was reproducibly detected at attomolar level. Some miRNA biomarkers for various cancers were found from FFPE samples.

(2) Serum and plasma are suggested to contain microsomes in which miRNA is enclosed. miRNA from serum and plasma samples were detected with high sensitivity and reproducibility with “3D-Gene”. Some miRNA biomarkers for various cancers were found from patients' serum.

“3D-Gene” is a potential tool for detecting transcriptome with high sensitivity and useful for quest and validation of diagnostic biomarkers from clinical samples.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-328. doi:10.1158/1538-7445.AM2011-LB-328
Abstract LB-333: Metabolic imaging of cancer using nanostructure-initiator mass spectrometry
Authors: Greving Matthew P. , Zhang Cathy , Yan Zhengming , Spilker Mary E. , Li Wenlin , Siuzdak Gary , O'Brien Peter J.
Published: July 2012

Metabolic adaptation is critical for the development and progression of cancer, and may influence the sensitivity of tumor cells to chemotherapy. Tumor metabolic profiling provides insights into those processes, but most quantitative methods are relatively limited in terms of assay sensitivity and spatial resolution, and by necessity focus on cultured cells, explanted tumors, or tissue extracts. Nanostructure-initiator Mass Spectrometry (NIMS) is a mass-based imaging technique that requires no sample preparation or extraction, and provides unbiased characterization of tissue metabolites in situ at low micron resolution. Seeking mechanistic insights and biomarkers of drug response, we used NIMS to profile xenobiotic- and cell-intrinsic metabolism in cultured Burkitt's lymphoma cells and breast cancer xenograft tumors treated with Rapamycin (RAPA) and Docetaxel (DOC), respectively. In both systems, non-radioactive fluorothymidine (FLT) metabolism was monitored as an indicator of cell proliferation, since FLT phosphorylation by thymidine kinase occurs mainly in G1-S phase cells. Rapamycin, FLT, and phosphorylated FLT metabolites were readily detected in Raji cells. RAPA, a starvation mimetic, induced dramatic alterations in metabolome of Raji cells 30, 60, and 90 minutes after treatment. Significant changes in cellular energetic- and biosynthetic intermediates were seen, including elevations in D-glucose (p=0.071); stearic acid (p=0.089); uridine monophosphate (p=0.002); N-Acetyl-L-glutamic acid (p=0.033); tiglyglycine (p=0.011); and isobutyryl carnitine (p=0.037). NIMS analysis of tumor tissues and normal liver from SCID-Beige mice implanted with MDA-MB-231 tumors showed FLT metabolite distribution consistent with that expected for proliferating cells. DOC induced derangement in that distribution and in endogenous metabolite profiles when compared to vehicle treatment. NIMS metabolomics thus allows simultaneous quantification of drug exposure, drug metabolism, and broad metabolic responses to treatment with single cell resolution. Metabolic discrimination of tissues, as with NIMS, is increasingly used to inform our understanding of pathology and to identify endogenous biomarkers. The approach described here, where differences in xenobiotic metabolism are used as landmarks of proliferating tumor cells in situ, offers novel tools for translating in vitro findings into a deeper understanding of tumor responses to therapy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-333. doi:10.1158/1538-7445.AM2011-LB-333