bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3964: Appropriate conditioning of CD4+T cells for antitumor immunotherapy.
Authors: Tanaka Tomohiro
Published: August 2014

It is now evident that lymphodepleting cytotoxic regimens augment antitumor immunity. We and others have demonstrated that transfer of naïve T cells after sublethal whole body irradiation induces antitumor effector T cells and inhibits tumor progression. We recently reported that CD4+CD25+Foxp3+ regulatory T cells (Treg) largely increased after whole body irradiation and suppressed the development of antitumor immune responses during recovery from lymphopenia. Depletion of these radio-resistant Treg following lymphodepletion significantly enhanced antitumor immunity (Baba J, et al. 2012, Blood). The combination of lymphodepletion and transfer of naïve T cells seems to be a promising approach for antitumor immunotherapy. But harvesting sufficient number of T lymphocytes from tumor-bearing patients is difficult. To obtain enough number of T cells that were capable of recognizing tumor-antigens, we expanded naïve T cells in vitro with culturing in CM containing several cytokines. CD4 or CD8 T cells from naïve spleens were activated with immobilized anti-CD3 mAbs for 2 days. Stimulated cells were maintained in CM with the combination of IL-2 (16 U/ml), IL-7 (10 ng/ml), IL-15 (10 ng/ml) and IL-23 (2 ng/ml) for additional 7 days. TCR-Vβ expressions of cultured T cells revealed that they maintained TCR diversity. To evaluate antitumor effects of these cells, mice were irradiated with 500 cGy to deplete lymphocytes and were transferred i.v. with the cultured T cells (4 x 107). On the same day, mice were inoculated s.c. with MCA205 tumor cells (1 x 105). We found that expanded CD4+ T cells mediated regression of s.c. tumors. In contrast, CD8+ T cells alone did not have therapeutic effects even combined with depletion of Treg. Next, we examined T cells proliferation by labeling with the CFSE and evaluated IFN-γ production after stimulation with specific tumor cells. Cultured CD4+ T cells proliferated rapidly and developed into tumor-specific effector T cells after transfer into lymphopenic hosts. Because CD4+ T cells from OT-II mice, that recognize Ovalbumin peptide specifically, did not show any antitumor effects after transfer into lymphopenic mice, this antitumor activity of CD4+ T cells is more likely to be tumor-antigen specific. Interestingly, using irradiated Rag-2 knockout mice as recipient abrogated antitumor effects of transferred cultured CD4+ T cells. Our results indicated that the combination of lymphodepletion and transfer of in vitro expanded CD4+ T cells has a potent antitumor efficacy, and recipient cells from irradiated hosts play a role in this treatment model.

Citation Format: Tomohiro Tanaka. Appropriate conditioning of CD4+ T cells for antitumor immunotherapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3964. doi:10.1158/1538-7445.AM2013-3964
Abstract 3966: Development of a fully-human chimeric antigen receptor against PSCA for the treatment of pancreatic cancer.
Authors: Abate-Daga Daniel , Lagisetty Kiran H. , Gattinoni Luca , Yu Zhiya , Restifo Nicholas P. , Rosenberg Steven A. , Morgan Richard A.
Published: August 2014

Pancreatic cancer represents the fourth leading cause of cancer-related death in developed countries. Despite remarkable advances in the understanding of its molecular pathophysiology, treatment options remain scarce, highlighting the need for novel therapies. Adoptive cell transfer (ACT) of lymphocytes engineered ex vivo to express chimeric antigen receptors (CAR) has come of age as a safe and clinically efficacious treatment for advanced cancers.

We sought to develop a CAR specific for prostate stem cell antigen (PSCA), a glycoprotein with restricted expression in normal tissues that is over-expressed in pancreatic cancer cells starting at early stages of malignant transformation.

In order to establish the optimal CAR design, we generated constructs containing antigen-recognition domains derived from mouse or human antibodies, and intracellular domains containing one or two co-stimulatory domains, in addition to CD3zeta. We compared multiple constructs and found that a CAR based on human monoclonal antibody Ha1-4.117 had greater reactivity in vitro. Using a murine model of ACT we observed that CAR-engineered human lymphocytes induced an antitumor effect on HPAC xenografts. Interestingly, unlike what has been described for other CARs, a second generation PSCA CAR (containing CD28 signaling domain) induced a more potent antitumor effect than its third generation counterpart (containing CD28 and 41BB domains).

Our results provide evidence to support PSCA as a target antigen for immunotherapy of pancreatic cancer, using adoptively transferred autologous lymphocytes engineered to express an Ha1-4.117-based CAR.

Citation Format: Daniel Abate-Daga, Kiran H. Lagisetty, Luca Gattinoni, Zhiya Yu, Nicholas P. Restifo, Steven A. Rosenberg, Richard A. Morgan. Development of a fully-human chimeric antigen receptor against PSCA for the treatment of pancreatic cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3966. doi:10.1158/1538-7445.AM2013-3966
Abstract 397: Increased Rho GTPase and reduced glycolysis activity in metastatic cancer cells.
Authors: Su Pei-Fen , Chang Shu-Hao , Ho Chia-Hsin , Huang Wei-Li
Published: August 2014

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer in the world. Metastases are resistant to conventional therapies and are responsible for 90% of tumour-related death. Better understanding on the molecular mechanism of metastasis may shed light on prevention and treatment of disease. We have established a systemic cellular model for metastases investigation. The parental SAS cells, a low tumorigenic HNSCC cell line; SASVO3 (VO3) cells, a highly tumorigenic cells derived from SAS; and SASVO3M-1 (M1) and SASVO3M-5 (M5) cells, two lines of pulmonary metastatic cells derived from SASVO3 cells using mice model. Transcriptome analysis revealed that M1 and M5 shared the most similar transcription signature and their common genes were designated common M. The profiles of VO3 and common M were distinct from that of parental SAS. Biological pathway analyses showed the signaling of cell movement such as integrin, RhoA, Cdc42, Rac, and actin polymerization were identified in metastatic cells. Of oncogenic properties, metastatic cells possess increased activities of foci formation, filopodia formation, wound healing, and in vivo pulmonary metastases. Interestingly, metabolism assay revealed that the glycolysis activity was increased in highly tumorigenic VO3 cells compare to parental SAS. Yet it was decreased in metastatic M5 cells compared to VO3 cells. “Reverse Warburg effect," which indicating a lowered glycolysis in tumor cells, has been demonstrated by others in metastatic breast cancer by in situ immunohistochemistry stain. The correlation of metabolism and metastasis of HNSCC cells will be discussed.

Citation Format: Pei-Fen Su, Shu-Hao Chang, Chia-Hsin Ho, Wei-Li Huang. Increased Rho GTPase and reduced glycolysis activity in metastatic cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 397. doi:10.1158/1538-7445.AM2013-397
Abstract 3979: In vitro analysis of pan-BCL-2 inhibitor GX15-070 (obatoclax) on human lymphocytes for the feasibility of combination immunotherapy.
Authors: Kim Peter , Hodge James , Grenga Italia , Donahue Renee , Schlom Jeffrey , Farsaci Benedetto
Published: August 2014

A previous murine study (B. Farsaci et al, Int. J. Cancer, 2010) showed that mice administered an anti-cancer vaccine prior to treatment with the pan-BCL-2 inhibitor obatoclax (GX15-070) showed a substantial reduction of metastatic mouse lung tumors due to increased apoptotic resistance of mature CD8T cells and decreased Treg function.

The purpose of this study was to evaluate activity of obatotclax on human lymphocyte populations. In vitro effects of obatoclax on human T cell subsets (naïve, central memory, effector-memory and terminal effector T cells) at different maturation states (CD69+ early-activated T cells and CD69- mature T cells) and regulatory T cells (Tregs) were evaluated at multiple concentrations (0.1 - 10 uM). Purified CD8 T cells or whole PBMCs from normal donors were activated for 3-4 days with anti-CD3 and anti-CD28 monoclonal antibodies (short-term culture) or maintained in an IL7/IL15-enriched medium for 7 additional days (long-term culture). After short- or long-term cultures, isolated CD8Ts and whole PBMCs were treated with obatoclax or control (DMSO) for 24 hours, and their viability and differentiation status were analyzed by flow cytometry.

The results indicate that (1) long-term cultured CD8 and CD4T cells were more resistant to obatoclax induced cell death than those in the short-term culture condition, (2) obatoclax increased the percentage of total memory CD8 and CD4T cells (central memory + effector memory) in the long-term culture condition, and (3) obatoclax increased the apoptosis of regulatory T cells (Tregs) and induced a profound down-regulation of FOXP3 in a dose dependent manner.

It is postulated that first, for an optimum combination effect, an immune-stimulating agent directed at T cells needs to precede obatoclax treatment long enough to allow T cells to acquire resistance to obatoclax. Then secondly, with proper treatment scheduling, memory T cells can be viably maintained, thereby establishing a sustainable source of cytotoxic T cells to kill tumors. Finally, because human regulatory T cells are sensitive to obatoclax, there is a potential for obatoclax to tip the numerical and functional balance of effector T cells and Tregs towards effector T cells in human tumor microenvironment. These conclusions support the preclinical murine in vivo study, and provide the rationale for combining obatoclax with an immunotherapeutic regimen in clinical studies.

Citation Format: Peter Kim, James Hodge, Italia Grenga, Renee Donahue, Jeffrey Schlom, Benedetto Farsaci. In vitro analysis of pan-BCL-2 inhibitor GX15-070 (obatoclax) on human lymphocytes for the feasibility of combination immunotherapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3979. doi:10.1158/1538-7445.AM2013-3979
Abstract 3982: Immuno-sensitization of malignant gliomas by natural polyphenols.
Authors: Mandalaparty Chandramouli , Huang Andrew , Mittal Sandeep , Rimando Agnes M. , Dou Q. Ping , Parajuli Prahlad
Published: August 2014

Malignant gliomas are the most common primary brain tumors with approximately 22,000 cases per year. In spite of aggressive surgery, radiotherapy and chemotherapeutic regimens, the median survival of patients with malignant gliomas is a dismal 15 months. The objective of our study was to explore the adjuvant therapeutic potential and associated molecular mechanisms of natural polyphenols stilbenes in malignant gliomas. Recent studies have demonstrated the efficacy of stilbenes in extracranial tumor models. Stilbenes, including resveratrol, were bioavailable in the brain even after low dose oral administration and there was no toxicity to normal tissue. On the contrary, these compounds were found to be neuroprotective in Alzheimer's disease models. Mechanistically, stilbenes modulate NF-κB, STAT3, wnt/β-catenin signaling and also inhibits antiapoptotic protein Bcl-xl. While these molecules are critical in imparting chemoresistance, they are also pivotal in inducing immune-resistance and pro-tumor immune infiltration in gliomas. We hypothesized that stilbenes enhance the efficacy of chemotherapy while also inhibiting immune-suppression and -resistance in malignant gliomas via overlapping molecular mechanisms. We observed significant positive interaction between Temozolomide and Resveratrol for inhibition of glioma proliferation. Resveratrol induced apoptosis in gliomas via inhibition of Bcl-2 and Bcl-xl, while STAT3 activity was also inhibited. Importantly, Resveratrol significantly enhanced the expression of TRAIL-R2 and MIC A/B in gliomas, making them vulnerable to lysis by CTLs and NK cells. Studies are underway to determine combined effect of DC-based immunotherapy and stilbenes against malignant gliomas using syngeneic, orthotopic mouse glioma models. The results can have significant impact in developing novel adjuvant studies to improve survival in patients with malignant gliomas.

Citation Format: Chandramouli Mandalaparty, Andrew Huang, Sandeep Mittal, Agnes M. Rimando, Q. Ping Dou, Prahlad Parajuli. Immuno-sensitization of malignant gliomas by natural polyphenols. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3982. doi:10.1158/1538-7445.AM2013-3982
Abstract 3988: Deregulated sphingosine 1-phosphate signaling contributes to a more aggressive phenotype in oral squamous cell carcinoma cells.
Authors: Johnson Steven , Yap Lee Fah , Narayanan Sathya , Paterson Ian
Published: August 2014

Oral squamous cell carcinoma (OSCC) is the sixth most common cancer worldwide, accounting for more than 250,000 new cases each year. Early death usually occurs as a result of local invasion and regional lymph node metastases and overall five year mortality rates are approximately 50%. Novel therapeutic targets are urgently required. Sphingosine 1-phosphate (S1P) regulates many biological processes associated with tumour development and progression, with the specific effects of S1P being determined, in part, by the profile of receptor expression in tumor cells. Sphingosine kinase 1 (SphK1), an enzyme responsible for the production of S1P, has been reported to be overexpressed in OSCC tissues, but the effect of S1P on the behavior of OSCC cells and the expression of S1P receptors in OSCC has not been examined. The effect of S1P on OSCC cells was examined by treating OSCC cell lines in vitro. S1P inhibited the proliferation of OSCC cells but stimulated cell scattering in colony dispersal assays, causing colonies to disperse into single, highly migratory cells. S1P-induced cell migration was dependent on the MEK, ROCK and Rac-1 signaling pathways. Additionally, S1P enhanced tumor cell invasion in 3-dimensional organotypic invasion assays. S1P also protected OSCC cells from Cisplatin-induced apoptosis, as determined in clonogenic survival assays. Using QPCR, we show that OSCC cell lines express S1P receptors 1-3 and that the expression of S1P1 and S1P2 was significantly lower in 8/8 OSCC cell lines compared to normal oral keratinocytes (p<0.001), whilst S1P3 mRNA levels were significantly higher in 6/8 cell lines (p<0.001). S1P receptor expression was similarly deregulated in two dysplasia cell lines, suggesting that alterations in S1P signalling occur early in tumor progression. The results of the present study demonstrate that de-regulated S1P signaling contributes to a more motile, invasive and chemo-resistant phenotype in OSCC and indicate that the S1P-receptor axis may represent a promising target for therapy. Work is ongoing to examine the protein expression of S1P receptors in OSCC tissues and to determine the precise role of the S1P3 receptor in mediating the tumor promoting effects of S1P in OSCC cells.

Citation Format: Steven Johnson, Lee Fah Yap, Sathya Narayanan, Ian Paterson. Deregulated sphingosine 1-phosphate signaling contributes to a more aggressive phenotype in oral squamous cell carcinoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3988. doi:10.1158/1538-7445.AM2013-3988
Abstract 3990: The role of Th17 cells in the microenvironment of oral squamous cell carcinoma.
Authors: Bu Rongfa
Published: August 2014

Objective: To find out if there is any abnormal in the Th17 level of OSCC patients in vivo, to affirm their inhibitory effects on OSCC and find relative mechanism.To find new way to control OSCC though Th-17 relatived pathway.

Methods: Flow cytometry was used to evaluate the level of Th17 and Treg in the peripheral blood samples of OSCC patients and control group. Th17 and Treg in The tumor microenvironment were detected too and the relation between them was evaluated. Stable transfection was performed to transfect human tongue carcinoma cell line TCA8113 with human-IL-17a gene. TCA8113/IL-17 cell and hu-PBL -SCID-beige mice were used to investigate the change of the tumor microenvironment and the possible mechanism of how Th17 inhibite the OSCC tumor grow.

Results: The level of Th17 cells in the peripheral blood of OSCC patients is higher than the normal control group; The level of CD4 + CD25high CD127lowT cell in peripheral blood of OSCC patients was higher than the normal control. The level of CD31+Treg in peripheral blood of OSCC patients was higher than the normal control. Th17 cells and Treg infiltration were found in the local tumor microenvironment of OSCC and a inverse relationship between them was found. The second part: TCA8113/IL-17 cell which stable express IL-17A was successfully established, and the shape was good. The proliferation of cells in vitro was samiliar to TCA8113 and TCA8113/PCDNA3.1 cells.The Hu-PBL-SCID-beige mice immune reconstitution model was successfully established, and human-IgG can be detected in peripheral blood of all mice with an average concentration of 2.105g/L. The success rate of reconstruction was 100%.Experimental mouse model of OSCC were constructed with TCA8113, TCA8113/IL-17, and TCA8113/pcDNA3.1 cell. Success rate was 95%.Tumor growth rate in T +17 group was lower than T and T+P group. The final tumor volume of T+17 group (1.219 ± 0.626cm3) was lower than T group (2.858 ± 1.188cm3) and T+P group (2.59±1.199 cm3).The mRNA and protein expression levels of IL-17a, IL-6, granzyme B and RORγt in T+17 group were higher than the T group and T + P group.

Conclusion:Th17 and Treg levels of peripheral blood were increased in OSCC patients, but there was no relation between them. The immune environment in OSCC patients was in a suppressive state. CD4 + CD25highCD127low was a better way than o CD4+CD25highFoxP3+ to marked Treg in OSCC. Infiltration of Th17 and Treg in tumor microenviroment could be evaluated, and negative correlation between them could be investigated. Treg in the OSCC microenvironment may inhibited the Th17 differentiation and function.

Citation Format: Rongfa Bu. The role of Th17 cells in the microenvironment of oral squamous cell carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3990. doi:10.1158/1538-7445.AM2013-3990
Abstract 4003: Influence of extracellular calcium on MAPK signalling cascade in colon cancer cells.
Authors: Tennakoon Samawansha , Schmid Johannes , Marian Brigitte , Kallay Enikö
Published: August 2014

Background: The calcium sensing receptor (CaSR) is a G-protein coupled receptor, which is capable of sensing even minute changes in extracellular calcium levels. The main role of the CaSR is regulation of calcium homeostasis. Besides the organs responsible for calcium homeostasis the receptor is expressed in a number of tissues including colonic epithelium and can be activated by several ligands, stimulating various downstream signalling pathways. Thus the CaSR is involved in numerous biological processes such as cell proliferation, differentiation and lipid membrane metabolism. Previous studies demonstrated that calcium regulates cell proliferation in colonocytes but the pathways or factors involved in this process still need to be elucidated.

Aim: The aim of this study is to investigate the impact of CaSR activation on the Mitogen-Activated Protein Kinase (MAPK) signalling cascade in colon cancer cell lines. We hypothesize that in colonocytes, activated CaSR down regulates proliferative pathways.

Methods: We studied the effect of CaSR ligands on downstream pathways through gene expression assays and protein analysis.

Results and Conclusion: We could show that the differentiation status of the cells (TC-7 a colon cancer cell line) determines whether a pathway is activated or rather inhibited by calcium treatment. Gene expression studies performed by luciferase assay and protein analysis by western blots reflect that extracellular calcium activated MAPK cascade in subconfluent TC-7 cells. On the other hand, short time (10 to 30 minutes) treatment of confluent/ differentiated cells with extracellular calcium showed no activation of ERK. Treatment for longer time (e.g 8h) inhibited ERK phosphorylation. The treatment of the cells with 100nM R-568, a calcimimetic that act only through the CaSR slightly inhibited ERK phosphorylation compared with the vehicle control, suggesting an anti-proliferative role of the CaSR.

Citation Format: Samawansha Tennakoon, Johannes Schmid, Brigitte Marian, Enikö Kallay. Influence of extracellular calcium on MAPK signalling cascade in colon cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4003. doi:10.1158/1538-7445.AM2013-4003
Abstract 4016: Rapamycin-resistant HIF-1α expression in hepatocellular carcinoma (HCC) cells and high sensitivity of endothelial cells within HCC tissues to rapamycin.
Authors: Tanaka Hiroki , Yamamoto Masahiro , Miyakoshi Masaaki , Ogawa Katsuhiro
Published: August 2014

HIF-1 plays a pivotal role in cancer development via cellular growth,proliferation, survival, metabolism and angiogenesis. High expression of HIF-1α, a regulatory component of HIF-1, in cancer cells is due to increased synthesis of HIF-1α via the increased translation of HIF-1α mRNA under the influence of activated mTORC1 signaling as well as decreased HIF-1α degradation that is either on oxygen-dependent or independent mechanism. In mouse hepatocarcinogenesis, our previous data demonstrated that HIF-1α is overexpressed depending on the PI3K-Akt pathway from earliest stage and that HIF-1α knockdown by using siRNA resulted in marked growth retardation and cell death in HCC cells. We here demonstrated that Akt, 4E binding protein 1 (4EBP1) and p70 ribosomal protein S6 kinase (S6K) were hyperphosphorylated in mouse hepatic tumors and HCC cells, indicating that the PI3K-Akt-mTORC1 pathway was activated. However, although rapamycin, an inhibitor of mTORC1, effectively suppressed phosphorylation of 4EBP1 and S6K in HCC cells, HCC cells were resistant to rapamycin where HIF-1α continued to be expressed during rapamycin treatment. This HIF-1α expression was due to de novo synthesis of HIF-1α during rapamycin treatment as well as HIF-1α stabilization due to binding of HIF-1α to HSP90 that was constitutively over-expressed in the HCC cells. On the other hand, rapamycin induced extensive hemorrhagic necrosis in HCC tissues in vivo but not in normal liver or adenomas (benign lesions). Phosphorylated ribosome protein subunit 6 (rps6), the downstream phosphorylation target of mTORC1-S6K pathway, was specifically detected in the endothelial cells within HCCs but not those in normal liver or adenomas. Furthermore, rapamycin decreased proliferation of endothelial cells in vitro, reducing HIF-1α expression together with the decreased 4EBP and S6K phosphorylation, indicating that the endothelial cells within HCCs were especially sensitive to rapamycin. Dual treatment with inhibitors for mTOR and HSP90, therefore, could target both HCC cells and endothelial cells within HCCs, which might be an effective therapy for HCCs.

Citation Format: Hiroki Tanaka, Masahiro Yamamoto, Masaaki Miyakoshi, Katsuhiro Ogawa. Rapamycin-resistant HIF-1α expression in hepatocellular carcinoma (HCC) cells and high sensitivity of endothelial cells within HCC tissues to rapamycin. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4016. doi:10.1158/1538-7445.AM2013-4016
Abstract 4021: Transcriptomic landscape of Ewing sarcoma based on RNAseq.
Authors: Zehir Ahmet , Ambati Srikanth R. , Moore Malcolm AS
Published: August 2014

Ewing sarcoma is the second most common primary bone tumor in children, constituting 3% of all pediatric tumors. The 5 year survival rate is 59% to 76% for children younger than 15 years and from 20% to 49% for those aged 15 to 19 years. 85% of Ewing sarcoma are characterized by a chromosomal translocation between the Ewing sarcoma gene (EWS) breaking point and the ETS family of transcription factor FLI1, which creates a new type of fusion transcription factor with novel functions. While EWS-FLI1 is the predominating translocation event, other types of translocations between EWS and other ETS transcription factors have been reported. Mesenchymal stem cells (hMSC) are thought to be the cell of origin for Ewing sarcoma. The molecular mechanisms of transformation of hMSCs with ectopic EWS-FLI1 expression has been extensively studied, however, a complete transformation of hMSCs into Ewing sarcoma tumor cells is not yet achieved suggesting there are other elements required for the transformation of hMSCs.

RNAseq has proved to be a powerful new method for capturing multiple arrays of information simultaneously such as gene expression, nucleotide variation, alternative splicing patterns and fusion transcripts. RNAseq has been used only once in the literature in identification of a novel fusion transcript in Ewing sarcoma tumor cells. Here we obtained RNAseq data from Ewing sarcoma patients and cell lines to gather novel insight into the pathogenesis of Ewing sarcoma. We obtained sequencing data from two Ewing sarcoma cell lines (A673 and SK-PN-DW); human brain vascular pericytes (HBVP), 8 patient tumors and 3 matched normal control tissues. Initial data analysis was performed through an in-house developed pipeline that uses publicly available software and further data analysis was performed using R with various packages. We describe the gene expression changes in patients compared to normal tissues as well as HBVP. We also discovered a group of novel fusion transcripts that we subsequently confirmed with RT-PCR. Since EWS is also an RNA splicing protein, changes in alternative splicing are also described.

Citation Format: Ahmet Zehir, Srikanth R. Ambati, Malcolm AS Moore. Transcriptomic landscape of Ewing sarcoma based on RNAseq. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4021. doi:10.1158/1538-7445.AM2013-4021
Abstract 4027: Pathway analysis of chemoresistance in ovarian cancer cell lines.
Authors: Jackson William P. , Katre Ashwini A. , Dobbin Zachary A. , Steg Adam D. , Landen Charles N.
Published: August 2014

Introduction: Ovarian Cancer is the most deadly female reproductive cancer and fifth most common cause of death from cancer among women. Limited characterization of the chemoresistant population demonstrates the need for improved therapy for ovarian cancer. Pathway analysis of microarray data of paired chemosensitive and chemoresistant ovarian cancer cell lines may help to elucidate important contributors to chemoresistance, and paired analyses can eliminate the significant heterogeneity among patient samples. Significant genes may serve as potential targets for novel drug therapies. Some genes likely contribute to resistance of multiple chemotherapies and represent common pathways of resistance.

Methods: Microarray data of three pairs of chemosensitive and chemoresistant ovarian cancer cell lines were analyzed using GeneSpring Single Experiment Analysis of 517 pathways. These pairs include SKOV3ip1 and SKOV3TRip2 (taxane-resistant), HeyA8 and HeyA8MDR (multi-drug resistant), and A2780cp20 and A780cp55 (increasing cisplatin resistance). Significant genes within significant pathways were compared between types of drug resistance.

Results: Between SKOV3ip1 and SKOV3TRip2, 8 pathways were significantly (p<0.001) modulated, dominated by the selenium pathway and folate/B12 metabolism. Between HeyA8 and HeyA8MDR, 9 pathways were significantly modified, led by DNA regulation and cell cycle progression. These differences between the SKOV3 model were interesting, given that both chemoresistant lines were developed in the same way. In A2780cp20 and A2780cp55, 7 pathways were significantly affected, highlighted by members of the Electron Transport Chain and controls on oxidative phosphorylation and autophagy. Validation of individual genes as important to chemoresistance will follow from this analysis.

Conclusions: Multiple pathways are implicated in chemoresistance, even when selected by the same chemotherapeutic agent. Pathways contributing to metabolism, DNA repair, cell cycle control, and stress responses dominated. The high variability in pathway overexpression highlights the importance of patient-specific approaches to overcoming chemoresistance in ovarian cancer.

Citation Format: William P. Jackson, Ashwini A. Katre, Zachary A. Dobbin, Adam D. Steg, Charles N. Landen. Pathway analysis of chemoresistance in ovarian cancer cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4027. doi:10.1158/1538-7445.AM2013-4027
Abstract 403: The role of HER2 in the brain metastatic cascade in breast cancer.
Authors: Liao Yunxiang , Osswald Matthias , Gril Brunilde , Steeg Patricia , Wick Wolfgang , Winkler Frank
Published: August 2014

Approximately 20% of brain metastases originate from breast cancer, which is the most common invasive cancer of women worldwide. HER2 inhibitors have revolutionized the treatment of HER2 positive breast cancer patients, but have no or marginal activity against brain metastases yet - which is becoming increasingly problematic, since 40% of HER2 positive breast cancer patients will eventually develop this devastating complication of the disease. Therefore we aimed to understand which step of the brain metastatic cascade is actually affected by HER2 overexpression; which is crucial for successful growth to a macrometastasis; and which is most amenable to a prophylactic therapy. For this we used our novel animal model which allows to non-invasively follow single metastasizing cancer cells through a chronic cranial window over time, using in vivo multiphoton microscopy. Human MDA-MB-231 breast cancer brain-seeking subclones with high HER2 expression or with control vector have been injected into the circulation of female nude mice. As shown before for other cancer entities, only a very small proportion of cancer cells initially arrested in brain capillaries can successfully form brain metastases, which was similar for both groups (1.02% vs 1.67% - for HER2 overexpression and vector control, respectively). However, more of the injected HER2+ cells managed to initially arrest compared with HER2- cells (47.5 vs. 27.7 per 100,000 injected cells). Moreover, they extravasated earlier, and showed more early interaction with brain capillaries leading to intensive vascular remodeling, but no angiogenesis. We therefore focussed on factors of early breast cancer cell - blood vessel - interaction, and found integrins to be overexpressed in HER2+ cells. In ongoing experiments, we test an integrin αvβ3-5 inhibitor regarding its activity to prevent the early metastatic steps, and find that these steps are markedly inhibitied in treated animals. Our results provide a better understanding of the breast cancer brain metastatic cascade, and the role of HER2. Importantly, they suggest that inhibition of crucial early steps involving cancer cell - blood vessel interactions via integrin pathways can serve as a well tolerated long-term prophylactic treatment for HER2-positive breast cancer patients which are at risk to develop brain metastases.

Citation Format: Yunxiang Liao, Matthias Osswald, Brunilde Gril, Patricia Steeg, Wolfgang Wick, Frank Winkler. The role of HER2 in the brain metastatic cascade in breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 403. doi:10.1158/1538-7445.AM2013-403
Abstract 4042: Selective inhibition of unfolded protein response induces apoptosis in pancreatic cancer cells.
Authors: Chien Wenwen , Sun Qiaoyang , Lim Su Lin , Varela Ana D S M , Sun Haibo , Gery Sigal , Koeffler H Phillip
Published: August 2014

Endoplasmic reticulum (ER) stress is associated with the proliferation of pancreatic tumor cells resulting from unfolded proteins. The unfolded protein response (UPR) is a signaling pathway activated to protect, restore, and aid cells in coping with ER stress. The direct correlation between pancreatic tumor cells and elevated UPR makes the many regulatory molecules of the pathway (e.g. IRE-1) critical targets for therapeutic approaches. The objective of our study was to assess potential therapeutic efficacy of inhibitors of UPR in pancreatic cancers and investigate the mechanisms underlying IRE-1 inhibitor-mediated pancreatic cancer apoptosis. Fourteen pancreatic cancer cell lines and one normal pancreatic epithelial cell line were studied. STF-083010 is a specific inhibitor of IRE-1, which is required for the splicing of XBP-1 mRNA. Spliced XBP-1 mRNA codes for a transcription factor that enhances transcription of chaperone proteins which reverse UPR. MTT assays showed a dose- and time-dependent growth inhibition of 8 pancreatic cancer cell lines by STF-083010, with IC50s ranging between 200 nM to 10 uM. Growth inhibition was also noted using a clonogenic growth assay in soft agar. Annexin V and cell cycle analysis showed that STF-083010 caused growth arrest at the G1 phase in 4 cell lines and induced cell apoptosis in 4 cell lines. Western blot analysis showed cleavage of caspase 3 and PARP, and the induction of the apoptotic molecule Bax. In addition, synergistic effects were found between STF-083010 and either gemcitabine or bortezomib. Our data suggest that an IRE-1 inhibitor is a novel therapeutic approach for treatment of pancreatic cancers.

Citation Format: Wenwen Chien, Qiaoyang Sun, Su Lin Lim, Ana D S M Varela, Haibo Sun, Sigal Gery, H Phillip Koeffler. Selective inhibition of unfolded protein response induces apoptosis in pancreatic cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4042. doi:10.1158/1538-7445.AM2013-4042
Abstract 4061: A novel CXCR4-mediated mechanism of docetaxel resistance in prostate cancer cells.
Authors: Bhardwaj Arun , Srivastava Sanjeev K. , Arora Sumit , Hyde Stephen J. , Andrews Joel F. , McClellan Steven , Singh Seema , Singh Ajay P.
Published: August 2014

Docetaxel (DTX), a semi-synthetic analog of paclitaxel, has emerged as the standard of care for chemotherapy of hormone-resistant prostate cancer. DTX confers its anti-neoplastic activity by inhibiting microtubule depolymerization, which leads to G2/M cell cycle arrest and subsequent apoptosis. However, most patients treated with DTX ultimately develop resistance to the drug and succumb to the disease. Therefore, understanding the mechanisms underlying DTX resistance is a priority area in prostate cancer research. Increasingly, it is being suggested that cancer cells may use common pathways for disease aggressiveness/metastatic spread and chemotherapeutic resistance. Therefore, we investigated the role of CXCL12/CXCR4 signaling, which is known to promote invasion and metastasis, in DTX-resistance of prostate cancer cells. Our data demonstrated that CXCL12 treatment rescued the prostate cancer cells from DTX-induced cytotoxicity and G2/M mitotic arrest. Furthermore, the cytoprotective effect of CXCL12 was abolished upon pretreatment of prostate cancer cells with AMD3100 (a small molecule antagonist of CXCR4) or siRNA-mediated silencing of CXCR4, thus confirming the role of CXCR4 in DTX-resistance. Immunofluorescence analyses revealed enhanced polymerization of microtubules in DTX-treated prostate cancer cells. In accordance with this, immunoblot analyses demonstrated increased levels of detyrosinated (glu-) and acetylated (ace-) tubulins, specific markers of the polymerized tubulin, in prostate cancer cells treated with DTX. Co-treatment of CXCL12 abrogated DTX-induced stabilization of microtubules in the prostate cancer cells, an effect that was diminished when the cells were pre-treated with AMD3100. Altogether, these initial findings demonstrate the role of CXCL12/CXCR4 signaling axis in DTX-resistance of prostate cancer cells and thus could be an attractive target for therapeutic enhancement of DTX.

Citation Format: Arun Bhardwaj, Sanjeev K. Srivastava, Sumit Arora, Stephen J. Hyde, Joel F. Andrews, Steven McClellan, Seema Singh, Ajay P. Singh. A novel CXCR4-mediated mechanism of docetaxel resistance in prostate cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4061. doi:10.1158/1538-7445.AM2013-4061
Abstract 4062: Clusterin downregulation sensitize prostate cancer cells to taxane by modulating mitosis.
Authors: Al Nakouzi Nader , Beraldi Eliana , Shepherd Stuart , Loriot Yohann , Kim Soojin , Leichmann Lauren , Zoubeidi Amina , Gleave Martin E
Published: August 2014

Clusterin (CLU) is a stress-activated molecular chaperone closely linked to treatment resistance and cancer progression. CLU is a novel therapeutic anti-cancer target with both preclinical and phase II clinical proof of concept using the antisense OGX-011 inhibitor; phase III clinical trials of combination docetaxel + OGX-011 are underway.

Cytotoxic chemotherapy drugs like taxane are believed to gain selectivity by targeting cells that are in mitosis. When cultured cancer cells are treated with taxane, only cells that enter mitosis are killed or rendered senescent. Quiescent cells or cycling cells that do not reach mitosis during drug exposure are spared. In this sense, sentisizing cells to taxane can occur through mitosis regulation.

In this study, we show that CLU downregulation enhances cytotoxicity of the new generation taxane cabazitaxol. Treatment of PC3 human prostate cancer cells with cabazitaxol induces G2/M arrest followed by mitotic death. Downregulation of CLU accelerated these G2/M associated events and resulted in reduced cabazitaxol EC50 and clonogenic survival upon cabazitaxol treatment. To investigate the mechanisms that allow CLU deficient cells to respond to taxane and examine whether this response is linked to a role of CLU in the control of cell cycle progression, we knocked down CLU expression in PC3 and LNCaP cells and found an accumulation of cells in G2/M phase and a reduction in cell growth. The screening of different cell cycle effectors after CLU downregulation in different cell lines and xenografts shows that CLU specifically regulates Cdc25C, while the two other Cdc25 isoforms, A and B, are not affected. Interestingly Cdc25C is a key regulator of mitosis initiation and exit. Importantly, we show that Cdc25C and CLU expression negatively correlate in prostate cancer cell lines, xenografts and human biopsies. When CLU is down regulated, Cdc25C transcription increased resulting in protein accumulation; as a consequence, cells showed G2/M blockage and slower mitotic progression. Accordingly, we suggest that down regulation of CLU alters sensitivity to taxane by modulating exit from mitosis, which is controlled by Cdc25C.

Citation Format: Nader Al Nakouzi, Eliana Beraldi, Stuart Shepherd, Yohann Loriot, Soojin Kim, Lauren Leichmann, Amina Zoubeidi, Martin E Gleave. Clusterin downregulation sensitize prostate cancer cells to taxane by modulating mitosis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4062. doi:10.1158/1538-7445.AM2013-4062
Abstract 4063: Histone methyltransferase G9a promotes the oral cancer cells recovery from drug-induced DNA damage.
Authors: Liu Chia-Wen , Cha Shih-Ting , Tan Ching-Ting , Kuo Min-Liang
Published: August 2014

Oral cancer is one of the serious life-threatening diseases in the world. In addition to surgery, the conventional treatment for oral cancer includes radiation and anti-cancer drugs, which usually act by induction of DNA damage. However, approximately one-third of treated patients will experience local or regional recurrence. G9a is a histone methyltransferase responsible for mono- or dimethylation of lysine 9 in histone H3. Our recent work has demonstrated that G9a level is highly correlated with aggressiveness and poor prognosis of lung, ovarian and colon cancer patients in Taiwanese population. Here, we report that knockdown of G9a resulted in decrease cell survival and cologenic capacity following DNA damage induced independently by etoposide and ionizing radiation. Depletion of G9a caused sustained phosphorylation of γ-H2AX after etoposide treatment. Together, our data suggest that a novel function of G9a may increase the DNA repair efficiency and promote the oral cancer cells recovery and survival from the DNA damage agents. We suggest that G9a may contribute to oral cancer proliferation by improvement of homologous recombination repair of DNA double break. Our study paves the way for exploring the blockade of G9a overexpression as a novel approach for the prevention and treatment of oral cancer.

Citation Format: Chia-Wen Liu, Shih-Ting Cha, Ching-Ting Tan, Min-Liang Kuo. Histone methyltransferase G9a promotes the oral cancer cells recovery from drug-induced DNA damage. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4063. doi:10.1158/1538-7445.AM2013-4063
Abstract 4069: FGFR2 expression and mutation are rare in papillary renal cell carcinoma.
Authors: Tsimafeyeu Ilya V. , Wynn Nigel , Gordiyev Marat , Khasanova Alfia
Published: August 2014

Papillary renal cell carcinoma (pRCC) is the second most common type of renal cell carcinoma (RCC). Fibroblast growth factor receptor (FGFR) 1 signaling has been implicated to play a role in RCC (Tsimafeyeu et al. ASCO meeting 2010). We evaluated expression and mutational activation of FGFR2 as a potential target for therapy of pRCC.

Formalin-fixed, paraffin-embedded specimens of removed 71 primary tumors from untreated pRCC patients were evaluated by immunohistochemistry with FGFR2 antibody. FGFR2 mutations were assessed by PCR and direct sequencing, with DNA obtained from 62 paraffin-embedded pRCC samples.

Expression of FGFR2 was observed in 8.5% of primary pRCC (6/71). Intensity was 3+ in all cases. 2 of 71 (3%) patients had nuclear FGFR2 expression. FGFR2 S252W mutation was detected at low frequency (1/62, 1.6%) in pRCC (type I). No N549K mutation was detected in pRCC (0/43).

FGFR2 expression and mutation are rare across papillary types of RCC.

This study was supported by Mr. Sergey Kartashov.

Citation Format: Ilya V. Tsimafeyeu, Nigel Wynn, Marat Gordiyev, Alfia Khasanova. FGFR2 expression and mutation are rare in papillary renal cell carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4069. doi:10.1158/1538-7445.AM2013-4069
Abstract 4108: SGEF is overexpressed in glioblastoma and mediates TWEAK-Fn14 induced cell survival.
Authors: Fortin Shannon P. , Mathews Ian T. , Rollo Jonathan , Ross Julianna T.D. , Symons Marc H. , Sarkaria Jann N. , Tran Nhan L.
Published: August 2014

Glioblastoma (GB) is the highest grade and most common form of primary adult brain tumors. Despite surgical removal followed by concomitant radiation and chemotherapy with the alkylating agent temozolomide (TMZ), GB tumors develop treatment resistance and recur, invading brain tissue. Impaired response to treatment occurs rapidly conferring a median survival of just fifteen months. Thus, it is necessary to identify the genetic and signaling mechanisms that promote tumor resistance and spread in order to develop targeted therapies to combat this refractory setting. Our lab has previously published a role for the TWEAK-Fn14 ligand-receptor system in GB cell invasiveness and survival. Here we report that SGEF, a RhoG specific guanine nucleotide exchange factor (GEF), is overexpressed in GB tumors and we hypothesize that SGEF plays an important role in promoting TWEAK-Fn14 mediated glioma invasion and survival. We show that upon TWEAK binding to Fn14, SGEF is recruited to the Fn14 cytoplasmic tail. The Fn14-SGEF interaction requires TRAF2 recruitment and leads to activation of Rac1. Importantly, SGEF mRNA expression is elevated in TMZ-resistant primary GB tumor grafts compared to those with known TMZ sensitivity. SGEF mRNA and protein expression are regulated by the TWEAK-Fn14 signaling axis in an NF-κB dependent manner and inhibition of SGEF expression via shRNA shutdown sensitizes glioma cells to TMZ-induced apoptosis and impairs colony formation following TMZ insult. Furthermore, gene expression analysis of SGEF depleted GB cells revealed impaired expression of a network of DNA repair and survival genes including ATM, ATR, BRCA2, and XIAP among others, as well as increased expression of the pro-apoptotic gene BAD. Understanding the role of SGEF in promoting chemotherapeutic resistance may direct the development of novel targeted therapeutics for TMZ refractory, invasive GB cells.

Citation Format: Shannon P. Fortin, Ian T. Mathews, Jonathan Rollo, Julianna T.D. Ross, Marc H. Symons, Jann N. Sarkaria, Nhan L. Tran. SGEF is overexpressed in glioblastoma and mediates TWEAK-Fn14 induced cell survival. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4108. doi:10.1158/1538-7445.AM2013-4108