bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1580: A novel C. elegans animal model for human RET receptor function.
Authors: Zelt Jordan D.S. , Chin-Sang Ian D. , Mulligan Lois M.
Published: August 2014

RET (Rearranged in Transfection) is a receptor tyrosine kinase mutated in the cancer syndrome multiple endocrine neoplasia 2 and linked to many other cancers, including medullary thyroid carcinoma and cancers of the adrenal gland. There are two distinct isoforms of RET, RET9 and RET51, with in vitro evidence indicating isoforms induce unique phosphorylation patterns on select tyrosine residues, possess different cell transforming and differentiation abilities, and bind/activate a unique set of substrates. However, these processes are challenging to dissect in complex organisms because both RET isoforms are ubiquitously co-expressed. We have generated a novel Caenorhabditis elegans model for RET expression to dissect the role of individual RET isoforms. C. elegans do not encompass a homologue of RET, thus we injected chimeric constructs in which human RET isoform sequences were fused to a N-terminal myristoylation signal for membrane targeting and expressed under the control of the C. elegans mechanosensory neuron-specific promoter, mec-4. The resultant transgenic animals were scored for phenotypes of RET-expressing neurons. We found that RET9 expressing animals have near WT phenotype, while RET51 expressing animals exhibited an axon termination phenotype where the PLM mechanosensory neuron of the tail fails to migrate to its correct location. This is indicative of early neuron terminal differentiation attributed to RET downstream signalling. Furthermore, strains harboring constitutively active oncogenic versions of RET9 or RET51 had severe PLM axon defects, with the RET51 phenotype being significantly more penetrant. Through generating strains with point mutations at select tyrosine residues and assessing the resulting phenotype, we have determined Y1062 and Y1096 to be main signalling hubs contributing to the RET-mediated phenotype in C. elegans. We are currently testing known kinase inhibitors, assessing efficacy and toxicity of potential RET inhibiting therapeutics. Using C. elegans as a platform to model RET is a quick and efficient system to gain insight on RET isoforms, delineate molecular events contributing to cancer progression, and investigate therapeutic agents that can be used to treat RET associated cancers.

Citation Format: Jordan D.S. Zelt, Ian D. Chin-Sang, Lois M. Mulligan. A novel C. elegans animal model for human RET receptor function. [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 1580. doi:10.1158/1538-7445.AM2013-1580
Abstract 1587: Loss of LZAP inactivates p53 in head and neck cancer and regulates sensitivity of cells to DNA damage in the p53-dependent manner.
Authors: Liu Dan , Issaeva Natalia , Yarbrough Wendell G.
Published: August 2014

We previously reported that tumor suppressor LZAP is lost in a portion of head and neck squamous cell carcinomas and that LZAP inhibits NF-kB, while activating p53 in ARF-dependent and -independent fashions. The ability of LZAP to regulate p53 in the absence of ARF has been largely unexplored. Here, we show that LZAP depletion diminished expression of mutant and wild-type p53 protein. LZAP activity toward p53 was independent of ARF and the known p53 regulators, Wip1 and HDM2. Loss of LZAP decreased p53 translation and destabilized p53 protein due, at least partially, to increased nucleolin expression. Knockdown of LZAP abrogated p53 stabilization induced by ionizing radiation, and LZAP depletion protected wild-type p53 cells from radiation or chemotherapeutic agents, while rendering cells expressing mutant or no p53 more sensitive. In human HNSCC, LZAP levels correlated with p53 levels, and remarkably, tumors with low LZ were less likely to have p53 mutations. These data suggest that loss of LZAP represents a novel mechanism of p53 inactivation in human cancer. Given these findings, inhibition of LZAP activity toward p53 for patients with tumors harboring p53 mutations may represent a therapeutic strategy to simultaneously sensitize tumors while protecting normal tissues from radiation or chemotherapy.

Citation Format: Dan Liu, Natalia Issaeva, Wendell G. Yarbrough. Loss of LZAP inactivates p53 in head and neck cancer and regulates sensitivity of cells to DNA damage in the p53-dependent manner. [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 1587. doi:10.1158/1538-7445.AM2013-1587
Abstract 1590: Sulfasalazine sensitizes glioblastoma cells to radiation treatment.
Authors: Sleire Linda , Skeie Bente S. , Netland Inger A. , Heggdal Jan , Pedersen Paal-Henning , Enger Per Ø.
Published: August 2014

Glioblastoma (GBM) is a lethal cancer with a limited response to ionizing radiation. Recent studies suggest that Sulfasalazine (SAS), a drug used to treat inflammatory bowel disease, inhibits the Xc− antiporter system in glioma cells, thereby blocking their uptake of cystein. Since the availability of cystein is a rate limiting step in intracellular antioxidant production, we wanted to investigate whether sulfasalazine sensitizes glioma cells to radiation.

Expression of xCT, the catalytic subunit of system Xc−, was found in 30 patient GBM biopsies. SAS effect on glioma cell growth was investigated using an electric cell substrate impedance sensing (ECIS) instrument. All glioma cell lines showed altered growth curves in response to SAS treatment. To assess the effect of blocking the antiporter, intracellular levels of the antioxidant glutathione were measured. With increasing doses of SAS, glutathione levels decreased in a dose response manner. In addition, cysteine was added to the medium to see if the toxic effects of SAS could be counteracted. Furthermore, accumulation of reactive oxygen species upon SAS treatment was measured. Glioma cells were also treated with escalating doses of SAS, alone or in combination with radiation (8 Gy). The presence of double stranded breaks increased markedly in the irradiated samples and also somewhat with increasing doses with SAS. In addition, cell death, viability and clonogenicity were investigated using live/dead staining, the MTS assay and the clonogenic assay.

All treatment groups exhibited increased rates of cell death compared to untreated controls. A combination of SAS and radiation resulted in higher levels of cell death, than radiation or SAS administered alone. Furthermore we continued with implantation of human GBMs into the brain of Nude rats. These animals were treated with Gamma Knife Radiosurgery alone or in combination with SAS. SAS were administered as a pre-treatment for three days before Radiosurgery. The rats receiving the combination treatment lived significantly longer compared to either treatment alone. Interestingly, the animals only receiving pre-treatment with SAS for three days lived significantly longer compared to the untreated controls, although this was not statistically significant. We are currently preparing a clinical trial for patients with GBM recurrences combining pre-treatment with Sulfasalazine and Gamma Knife Radiosurgery.

Citation Format: Linda Sleire, Bente S. Skeie, Inger A. Netland, Jan Heggdal, Paal-Henning Pedersen, Per Ø. Enger. Sulfasalazine sensitizes glioblastoma cells to radiation treatment. [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 1590. doi:10.1158/1538-7445.AM2013-1590
Abstract 1594: Sustained release of drug eluting nanoparticles from implantable devices for loco-regional chemoradiation therapy.
Authors: Kumar Rajiv , Belz Jodi , Markovic Stacey , Jadhav Tej , Nguyen Paul , Niedre Mark , DAmico Anthony , Makrigiorgos Mike , Cormack Robert , Sridhar Srinivas
Published: August 2014

Systemic chemotherapy is often used with radiation therapy in the management of prostate, cervix and lung cancer patients, but leads to severe systemic toxicities. We have introduced a new modality of loco-regional chemoradiation therapy termed in-situ image guided radiation therapy (BIS-IGRT) that offers the potential to deliver planned, localized and sustained delivery of chemotherapy agent, without systemic toxicities, as part of routine minimally invasive image guided radiation therapy procedures. Such image guided chemoradiation therapy requires characterization of the drug distribution produced by implantable drug eluters. This work presents imaging based means to measure temporal and spatial properties of diffusion distributions around spacers coated with dye-loaded nanoparticles.

The distribution of 250nm silica nanoparticles (NP) conjugated to Cyanine 7.5 dye was evaluated with a custom built high-speed near-infrared small animal imaging platform providing 0.1 millimeter spatial resolution with >1Hz image acquisition rate. A brachytherapy spacer loaded with the Cy7.5/silica NP was injected subcutaneously on the left hind flank of a mouse and one was inserted into a xenograft tumor on the opposite flank as part of an approved animal research protocol. The mouse was imaged more than 36 days.

The in vivo imaging experiments show that the area of high signal increases with time suggesting that NP accumulate in the vicinity of a spacer without diffusing to the rest of the body. The spatial and temporal characteristics of NP accumulation indicate that a BIS-IGRT approach may provide an effective means to improve the therapeutic ratio of brachytherapy.

Conclusion: In-vivo measurements demonstrate that NP remain resident in the vicinity of the implanted eluting spacers with accumulation over times appropriate to improve brachytherapy's therapeutic ratio. Future work will optimize the NP and substrate properties of an implantable spacer to attain the optimal chemotherapy distributions for simultaneous placement during image guided brachytherapy implants.

We acknowledge partial support from NSF DGE 0965843 and HHS/5U54CA151881-02.

Citation Format: Rajiv Kumar, Jodi Belz, Stacey Markovic, Tej Jadhav, Paul Nguyen, Mark Niedre, Anthony DAmico, Mike Makrigiorgos, Robert Cormack, Srinivas Sridhar. Sustained release of drug eluting nanoparticles from implantable devices for loco-regional chemoradiation therapy. [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 1594. doi:10.1158/1538-7445.AM2013-1594
Abstract 1597: C646, a selective small molecule inhibitor of p300, radiosensitizes cancer cells through enhancement of mitotic catastrophe.
Authors: Oike Takahiro , Komachi Mayumi , Ogiwara Hideaki , Yokota Jun , Nakano Takashi , Kohno Takashi
Published: August 2014

Background: The histone acetyltransferases (HATs) including p300, CBP and TIP60 are transcriptional co-activators implicated in many gene regulatory pathways and protein acetylation events. We have previously shown that siRNA-mediated ablation of p300 and CBP sensitized cancer cells to ionizing radiation (IR) (Ogiwara, Oncogene 2011), and that garcinol, curcumin and anacardic acid, natural compounds with multi-HATs inhibitory activity, had radiosensitizing effect in cancer cells (Oike, Int J Radiat Oncol Biol Phys 2012). However, radiosensitization by selective small molecule inhibitors targeting activity of a specific HAT, that can have much potential for clinical use, have not been well investigated. C646 is a selective small molecule inhibitor of p300 identified by structure-based and active site-directed in silico screening (Bowers, Chem Biol 2010). In this study, we investigated the in vitro effect of C646 on radiosensitization and cell death in human cancer cells.

Materials and Methods: The radiosensitizing effect in A549 (lung adenocarcinoma), H460 (lung squamous cell carcinoma) and H157 (lung large cell carcinoma) cells were assessed by colony formation assay, while that in MRC-5 (fibroblast) cells lacking in colony-forming ability was examined by ATP assay. Cell cycle distribution in A549 cells was analyzed using flow cytometry. The effect on mitotic catastrophe, apoptosis and senescence in A549 cells was evaluated by DAPI, annexin V and senescence-associated β-galactosidase staining, respectively. C646 was used at concentrations less than IC50.

Results: C646 radiosensitized A549, H460 and H157 cancer cells with dose enhancement ratio at 10% surviving fraction of 1.4, 1.2 and 1.2, respectively. Meanwhile, C646 unaffected the radiosensitivity of MRC-5 cells. Cell cycle analysis showed that the hyperploid cell population (> 4N DNA cells) increased in C646-treated A549 cells after IR. Analyses of mode of cell death revealed that C646 enhanced mitotic catastrophe, but not apoptosis nor senescence, induced by IR in A549 cells.

Conclusion: These data indicate that C646 is a radiosensitizer which enhances mitotic catastrophe induced by IR.

Citation Format: Takahiro Oike, Mayumi Komachi, Hideaki Ogiwara, Jun Yokota, Takashi Nakano, Takashi Kohno. C646, a selective small molecule inhibitor of p300, radiosensitizes cancer cells through enhancement of mitotic catastrophe. [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 1597. doi:10.1158/1538-7445.AM2013-1597
Abstract 1606: Photochemical internalization (PCI): a novel technology for site-specific drug delivery.
Authors: Berg Kristian , Hopper Colin , Weyergang Anette , Selbo Pål K. , Høgset Anders
Published: August 2014

The utilization of macromolecules in therapy of cancer and other diseases is becoming increasingly important. Recent advances in molecular biology and biotechnology have made it possible to improve targeting and design of cytotoxic agents, DNA complexes and other macromolecules for clinical applications. To achieve the expected biological effect of these macromolecules in many cases internalisation to the cell cytosol is crucial. At an intracellular level, the most fundamental obstruction for cytosolic delivery of therapeutic macromolecule is the membrane-barrier of the endocytic vesicles. Photochemical internalisation (PCI) is a novel technology for release of endocytosed macromolecules into the cytosol. The technology is based on the use of photosensitizers located in endocytic vesicles that upon activation by light induces rupture of the endocytic vesicles and thereby release of the macromolecules into the cytosol. PCI has been shown to enhance the biological activity of a large variety of macromolecules and other molecules that do not readily penetrate the plasma membrane, including type I ribosome-inactivating proteins (RIPs), gene-encoding plasmids, adenovirus, oligonucleotides and the chemotherapeutic agent bleomycin. For clinical utilization a novel photosensitizer has been developed and evaluated for PCI of bleomycin. Preclinical results with this combination will be presented. Furthermore, a phase I/II dose-escalating clinical trial has been finalized and results from this trial will also be presented.

Citation Format: Kristian Berg, Colin Hopper, Anette Weyergang, Pål K. Selbo, Anders Høgset. Photochemical internalization (PCI): a novel technology for site-specific drug delivery. [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 1606. doi:10.1158/1538-7445.AM2013-1606
Abstract 1609: IRF1 regulates bevacizumab induced autophagy .
Authors: Liang Ji , Piao Yuji , de Groot John F.
Published: August 2014

Antiangiogenic therapy is commonly being used for the treatment of recurrent glioblastoma. However, the benefits of angiogenesis inhibitors are typically transient and resistance often develops. Determining the mechanism of treatment failure of the VEGF monoclonal antibody bevacizumab for malignant glioma would provide insight into approaches to overcome therapeutic resistance. In this study, we observed up-regulation of autophagy marker expression and increased autophagosome formation after bevacizumab treatment. In order to determine genes regulating bevacizumab resistance, microarray gene expression analysis was performed on murine xenograft models of glioblastoma. Compared to control tumor tissues, we found increased transcriptional levels of STAT1/IRF1 signaling in bevacizumab resistant tumors, which were further confirmed by immunohistochemistry of bevacizumab treated tissue at the protein level. To determine whether IRF1 contributes to bevacizumab resistance, glioma stem cell lines with stable IRF1 down regulation were injected into the brains of nude mice. H&E staining of

6 week treated tumor tissues showed that bevacizumab markedly reduce tumor size in the shIRF1 group compared to the control shRNA group treated with bevacizumab. We also found a decrease in autophagy marker expression and increased TUNEL staining in shIRF1 cells. These data suggest that IRF1 may regulate bevacizumab-induced autophagy, and may be one important mediator of bevacizumab resistance.

Citation Format: Ji Liang, Yuji Piao, John F. de Groot. IRF1 regulates bevacizumab induced autophagy . [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 1609. doi:10.1158/1538-7445.AM2013-1609
Abstract 1611: Transcriptome analyses of the tumor cell line resistant to anti-VEGF therapy.
Authors: Li Dong , Xie Kun , Ding Guitao , Li Hongwen , Yao Xuejing , Jiang Cizhong , Fang Jianmin
Published: August 2014

Despite overt clinical success of anti-VEGF (vascular endothelial growth factor) agents, such as Avastin, for cancer patients, increasing evidence shows that prolonged treatment of anti-VEGF therapy results in tumor resistance. In order to understand how tumor cells become resistant to anti-VEGF therapy, we developed a mouse Lewis lung carcinoma (LLC) cell line, which was resistant to a potent VEGF blocker, VEGF-Trap. The cell line was established through five rounds of VEGF-Trap treatment and selection in vivo. In each round, C57BL/6 mice were inoculated with LLC cells and subjected to VEGF-Trap or PBS treatment. Tumors grown from treated mice were harvested and the isolated cells were injected to a new batch of mice for the next round of treatment. It was found that tumor cells became insensitive to VEGF-Trap in vivo after five rounds of VEGF-Trap treatment, i.e., VEGF-Trap no longer inhibited tumor growth. In contrast, VEGF-Trap remained efficacious to the LLC cells which underwent five passages in mice but treated with PBS. The resistance phenotype was maintained in LLC cells after the tumor cells were expanded for a number of passages in vitro, indicating that the resistance was due to inherited alterations in tumor cells. To gain insights into the molecular mechanism underlying the resistance to anti-VEGF therapy, we compared transcriptome profiles of the resistant and non-resistant tumor cells using mRNA-seq. The next generation sequencing reads were aligned to mouse genome; gene transcripts were assembled and calculated for relative abundance for individual gene. Results showed that 644 genes were differentially expressed with statistically significances, 317 up-regulated and 327 down-regulated in the resistant tumor cells. Interestingly, VEGF-C was significantly up-regulated in the resistant tumor cells (fold change = 4.91), which was further verified by real-time PCR analysis. Given the stimulatory effects of VEGF-C on both angiogenesis and lymphatic angiogenesis, it is possible that VEGF-C over-expression plays a role in the development of LLC tumor resistance to anti-VEGF therapy.

Citation Format: Dong Li, Kun Xie, Guitao Ding, Hongwen Li, Xuejing Yao, Cizhong Jiang, Jianmin Fang. Transcriptome analyses of the tumor cell line resistant to anti-VEGF therapy. [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 1611. doi:10.1158/1538-7445.AM2013-1611
Abstract 1612: Targeting intercellular adhesion molecule-1 (ICAM-1) prolongs glioblastoma survival in combination with bevacizumab .
Authors: Piao Yuji , Liang Ji , Henry Verlene , Holmes Lindsay , de Groot John F.
Published: August 2014

Glioblastoma is the most common malignant brain tumor and is characterized by cellular heterogeneity, vascular proliferation and extensive tissue infiltration.

Analysis of gene expression data from orthotopic glioma stem cells with acquired resistance to bevacizumab therapy identified over expression of intercellular adhesion molecule 1 (ICAM1, CD54) in resistant tumors. ICAM1 is a cell adhesion glycoprotein of the immunoglobulin supergene family which interacts with β2 integrins, mediates leukocyte transendothelial migration and T cell activation during inflammatory processes.

ICAM1 over expression in tumors resistant to antiangiogenic therapy was validated by real time PCR and Western blot. In a panel of glioma stem cell lines, ICAM1 expression was higher in mesenchymal compared to proneural cell lines.

GFP-tagged ICAM1 shRNA lentivirus was used to knockdown ICAM1 in multiple cell lines. We injected shICAM1 NSC11 and scramble glioma stem cells into the brain of nude mice. Mice bearing tumors formed from shICAM1 NSC11 cells survived significantly longer than mice injected with scramble control cells. Tumor size was significantly decreased in mice bearing tumors formed from shICAM1 cells than that in mice bearing tumors from GFP-tagged NSC11 control cells. Knocking-down ICAM1 suppresses tumor invasion both in vitro and in vivo. We next examined the mechanism of ICAM1 over expression after treated with anti-VEGF therapy. We observed that ICAM1 over expression correlated with hypoxia-inducible factor by immnofluorescence analysis in tumor tissue. Furthermore, we found that ICAM1 protein expression was markedly increased after NSC11 and NSC17 cells under hypoxic condition in a time-dependent manner. Furthermore, hypoxia-induced p-STAT3 increased transcription of ICAM1, which was inhibited by an inhibitor of the JAK/STAT3 pathway.

Our studies identify ICAM1 as a potentially important mediator of tumor migration/invasion in glioblastoma. Targeting ICAM1 may provide a new strategy to prolong the efficacy of antiangiogenic therapy and prevent the invasive phenotype.

Citation Format: Yuji Piao, Ji Liang, Verlene Henry, Lindsay Holmes, John F. de Groot. Targeting intercellular adhesion molecule-1 (ICAM-1) prolongs glioblastoma survival in combination with bevacizumab . [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 1612. doi:10.1158/1538-7445.AM2013-1612
Abstract 1616: Survivin regulates endothelial cell proliferation, survival and angiogenesis.
Authors: Eberlein Cath , Opoku-Ansah Grace , Legg James , London Tim , Gebhardt Frank , Davies Gareth , Tigue Natalie , McCourt Matthew , White Julia , Cross Darren , Schaeper Ute , Valge-Archer Viia E.
Published: August 2014

Background - Survivin is a member of the inhibitor of apoptosis protein (IAP) family, and is reported to be overexpressed in a number of human cancers with high unmet medical need including breast, non-small-cell lung (NSCLC), colorectal, gastric, glioblastoma, and pancreatic cancers. Multiple studies have described survivin as a key regulator of tumour cell proliferation, survival and drug resistance, which has led to the advancement of survivin inhibitors, including antisense and small molecule drugs, and cancer vaccines, into clinical testing. In the tumour micro-environment, survivin overexpression has also been reported in tumour blood vessels and conditional deletion of survivin in endothelial cells results in murine embryonic lethality secondary to haemorrhage and defective vascular development. We therefore hypothesised that inhibition of survivin function in endothelial cells could have anti-angiogenic or vascular disruptive effects.

Methods and Results - To better understand the role of survivin in primary endothelial cells we designed siRNAs capable of targeting multiple isoforms of human survivin. Using high content imaging assays we demonstrated that the siRNA were highly selective for survivin, inhibited the expression of multiple survivin splice variants, and had potent in vitro effects on endothelial cells. Survivin knockdown resulted in inhibition of cell cycle progression with the formation of enlarged, multi-nucleated cells, and in reduced endothelial cell survival. Survivin knockdown also impaired the formation of tube-like networks in vitro when co-cultured with human fibroblasts, but did not disrupt more established networks.

Conclusion These data show an important role for survivin in primary endothelial cells, and suggest that targeted inhibition of survivin may have anti-angiogenic effects, but may not disrupt more established blood vessels.

Citation Format: Cath Eberlein, Grace Opoku-Ansah, James Legg, Tim London, Frank Gebhardt, Gareth Davies, Natalie Tigue, Matthew McCourt, Julia White, Darren Cross, Ute Schaeper, Viia E. Valge-Archer. Survivin regulates endothelial cell proliferation, survival and angiogenesis. [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 1616. doi:10.1158/1538-7445.AM2013-1616
Abstract 1622: Dovitinib preferentially targets endothelial cells rather than cancer cells for the inhibition of hepatocellular carcinoma growth and metastasis.
Authors: Chen Zhi-Yuan , Shi Ming , Peng Li-Xia , Wei Wei , Li Xin-Jian , Guo Zhi-Xing , Li Shu-Hong , Zhong Chong , Qian Chao-Nan , Guo Rong-Ping
Published: August 2014

Background: Dovitinib is a receptor tyrosine kinase (RTK) inhibitor targeting vascular endothelial growth factor receptors, fibroblast growth factor receptors and platelet-derived growth factor receptor β. Dovitinib is currently in clinical trials for the treatment of hepatocellular carcinoma (HCC), however, its cellular target is not clarified.

Method: In this study, we used five HCC cell lines and five endothelial cell lines to validate molecular and cellular targets of dovitinib.

Results: Tumor growth and pulmonary metastasis were significantly suppressed in an orthotopic HCC model. Immunoblotting revealed that among known dovitinib targets, only PDGFR-β was expressed in two HCC cell lines, while four of five endothelial lines expressed PDGFR-β, FGFR-1, and VEGFR-2. Dovitinib inhibited endothelial cell proliferation and motility at 0.05 μmol/L, a pharmacologically relevant concentration; it was unable to inhibit the proliferation or motility of HCC cells at the same concentration. Immunohistochemical analyses showed that dovitinib significantly decreased the microvessel density of xenograft tumors, inhibiting proliferation and inducing apoptosis in HCC cells.

Conclusion: Our findings indicate that dovitinib inhibits HCC growth and metastasis preferentially through an antiangiogenic mechanism, not through direct targeting of HCC cells.

Citation Format: Zhi-Yuan Chen, Ming Shi, Li-Xia Peng, Wei Wei, Xin-Jian Li, Zhi-Xing Guo, Shu-Hong Li, Chong Zhong, Chao-Nan Qian, Rong-Ping Guo. Dovitinib preferentially targets endothelial cells rather than cancer cells for the inhibition of hepatocellular carcinoma growth and metastasis. [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 1622. doi:10.1158/1538-7445.AM2013-1622
Abstract 1628: Molecular cross-talk between head and neck squamous cell carcinoma cells and cancer-associated fibroblasts.
Authors: Ansell Anna , Kankainen Matti , Monni Outi , Johansson Ann-Charlotte
Published: August 2014

One of the main components of the tumor stroma is the cancer-associated fibroblasts (CAFs), which are known to stimulate tumor cell proliferation, angiogenesis and metastasis. Increasing evidence suggests that CAFs also influence the tumor cell response to various treatments. In this study we aimed to characterize the molecular cross-talk between CAFs and head and neck squamous cell carcinoma (HNSCC) cells. Tumor cell lines and CAF cultures were established from tumor biopsies derived from five HNSCC patients at Linköping University Hospital. Patient-matched tumor cells and CAFs were co-cultured for seven days in cell culture flasks, allowing cell-cell contact. After cell sorting the gene expression in mono-cultured and co-cultured tumor cells was investigated by Affymetrix microarray. Molecular networks were analyzed by Ingenuity Pathway Analysis (IPA). The expression of selected genes was verified by quantitative real-time PCR (qPCR) analysis. Fiftyeight genes were found to be differentially expressed (q<0.05) in tumor cells co-cultured with CAFs when compared to tumor cells in mono-culture. The top functions of these genes were cancer, cellular movement, and embryonic development as analyzed by IPA. Four genes (POSTN, GREM1, COL1A2, and COL6A3) were upregulated by more than 2-fold while 14 genes were found to be downregulated by at least 50%. The four upregulated genes have all been associated with epithelial-to-mesenchymal transition (EMT), increased migration, and TGFβ-pathway signaling. By qPCR analysis the expression of POSTN, GREM1 and COL1A2 was found to be induced by several hundred-fold in some of the cell lines under co-culture conditions. There were no significant changes in the expression of these genes when tumor cells and CAFs were co-cultured in a trans-well system, suggesting that the effect may be contact-dependent. Functional analyses are now being performed to determine the importance of these findings.

Citation Format: Anna Ansell, Matti Kankainen, Outi Monni, Ann-Charlotte Johansson. Molecular cross-talk between head and neck squamous cell carcinoma cells and cancer-associated fibroblasts. [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 1628. doi:10.1158/1538-7445.AM2013-1628
Abstract 1640: Combined inhibition of EMMPRIN and EGFR abrogates growth and migration of head and neck squamous cell carcinoma cells.
Authors: Suzuki Shinsuke
Published: August 2014

Head and neck cancer squamous cell carcinoma (HNSCC) is the most common malignancy of the upper aerodigestive tract. Despite intense efforts to improve different treatment modalities, mortality rates in advanced cases remain high. A deeper understanding of the HNSCC progression mechanisms is needed to improve survival in patients with HNSCC. The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase, which regulates crucial cellular signaling pathways contributing to tumor progression. EGFR is frequently amplified and overexpressed in several human solid tumors including a high percentage of HNSCCs. Evidence accumulated in several studies has shown that EGFR stimulation induces expression of matrix metalloproteinases (MMPs), which degrade extracellular matrix, and its activation process is critical for tumor development. Recently, extracellular matrix metalloproteinase inducer (EMMPRIN), which is known as CD147 and is a member of the immunoglobulin superfamily, has been reported to be highly expressed in malignant tumors including HNSCC. It has been reported that EGFR expression is associated with EMMPRIN in some solid tumors; however, the relationship of EMMPRIN with EGFR in head and neck cancers is not fully understood. To determine the relationship between EMMPRIN and EGFR in HNSCC, HNSCC cells were stimulated with epidermal growth factor (EGF), a ligand of EGFR. Immunoblotting analysis showed that EMMPRIN expression in HNSCC cells was upregulated by EGF in a dose- and time-dependent manner. In addition, gelatin zymography showed that EGF stimulation-induced cell invasion and MMP-9 expression of HNSCC cells. Interestingly, this increase in invasion and MMP-9 expression were abrogated by downmodulation of EMMPRIN expression using the siRNA technique. Furthermore, to determine the effects of combined EMMPRIN and EGFR targeting in the HNSCC cell line, HNSCC cells were investigated in cell proliferation and migration assays. The effects of combined treatment with the EMMPRIN function blocking antibody and the EGFR inhibitor AG1478 were examined on the proliferation and migration of HNSCC cells. This combined treatment resulted in greater inhibition of HNSCC cell proliferation and migration compared with the individual agents alone. These results suggest that EMMPRIN mediates EGFR-induced invasion and MMP-9 expression in cases where combined targeting of EMMPRIN and EGFR may be an efficacious treatment approach.

Citation Format: Shinsuke Suzuki. Combined inhibition of EMMPRIN and EGFR abrogates growth and migration of head and neck 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 1640. doi:10.1158/1538-7445.AM2013-1640
Abstract 1643: Physical and functional interactions between acid sensing ion channel 1 (ASIC1) and integrin β1 in glioma cells.
Authors: Rooj Arun K. , McNicholas Carmel M. , Fuller Catherine M.
Published: August 2014

The local microenvironment of a cancer cell plays important roles in tumor metastasis. Major components of this local niche are ion channels, integrin receptors, and the extracellular matrix.

Glioma cells express a specific amiloride-sensitive and constitutively active non-selective cation channel composed of ASIC1, α- and gENaC subunits, members of the Deg/ENaC superfamily of non-voltage gated sodium channels. Inhibition of this channel is associated with reduced migration and proliferation of the glioma cell. As gliomas are highly invasive, we hypothesized that channel activity was engaging an intracellular signaling pathway regulating migration and proliferation by interacting with integrin β1. We have shown the physical proximity of the ASIC1 subunit and integrin β1 by co-immunoprecipitation in D54MG glioma cells. A functional interaction between these two proteins was demonstrated when we found that stable (shRNA) knock-down of integrin β1 attenuated the amiloride-sensitive current by 89±24% (n≥9; ±SD P<0.001). Furthermore, prolonged (24h) inhibition of this glioma channel by PcTX-1, a specific ASIC1 inhibitor, down-regulated integrin β1 expression (by 60% ± 12% (SD) n≥3 P<0.001).

The surface expression of ASIC1 in glioma cells is also regulated by integrin β1. Using biotinylation, we found that stably knocking down integrin β1 inhibited the surface expression of ASIC1. In contrast, up-regulating membrane expression of integrin β1 by coating dishes with either fibronectin or vitronectin, increased the surface expression of ASIC1. This effect was not observed when dishes were coated with poly-L-lysine. We have previously shown that knockdown of ASIC1 reduced the phosphorylation level of ERK1/2, a key mediator of tumor cell proliferation and migration (Rooj et al. J. Biol. Chem. 287:4053, 2012). In the present study, phosphorylation of ERK1/2 was also reduced by knockdown of integrin β1 (by 58% ± 20% (SD) n≥3 P<0.001). Our data reveal a novel interaction between ASIC1 and integrin β1 in the regulation of the MAPK pathway that may form a mechanism by which changes in Na+ current can modulate cell proliferation and migration.

Citation Format: Arun K. Rooj, Carmel M. McNicholas, Catherine M. Fuller. Physical and functional interactions between acid sensing ion channel 1 (ASIC1) and integrin β1 in glioma 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 1643. doi:10.1158/1538-7445.AM2013-1643
Abstract 1644: Microglia promote glioma cell migration and dispersal by activating the PLCγ1 and Pyk2 signaling cascade.
Authors: Rolon-Reyes Kimberleve , Mendez Miguel , Rivera-Aponte David E. , Skatchkov Serguei N. , Eaton Misty J. , Kucheryavykh Lilia
Published: August 2014

Glioblastoma multiforme is one of the most aggressive and fatal brain cancers mostly because of the highly invasive nature of glioma cells. The ability of glioma cells to disperse to healthy brain stroma makes surgical and chemotherapeutic treatments mostly ineffective. Microglia infiltrate most gliomas and release factors, which favor tumor growth and dispersal. We hypothesize that microglia residing within the tumor stimulate glioma cell migration and dispersal through the phospholipase C (PLC)γ1 and proline rich tyrosine kinase 2 (Pyk2) signaling cascade. In the present study, we investigated rodent C6 and GL261 as well as human glioma cell lines with varying levels of invasiveness: U-87MG, A172, and HS683. Using a standard invasion assay, we demonstrated that the presence of rodent or immortalized human microglia in the bottom compartment of Boyden chambers activated invasion of all glioma cell types. Knock-down of PLCγ1 and Pyk2 proteins using siRNA reversed the stimulatory effect of microglia on glioma invasion in both cell lines. Pharmacological blockers of PLC (U73872 1nM) and Pyk2/focal adhesion kinase (FAK) (PF562271 20nM) also completely eliminated the ability of microglia to stimulate glioma cell migration in a wound healing assay. Using Western blot, we found upregulation of phosphorylated Tyr579/580 Pyk2 protein in glioma cells treated with microglia conditioned medium. Taken together, these data indicate that microglial cells activate glioma cell migration/dispersal through the pro-migratory Pyk2 and PLCγ1 signaling pathway in glioma cells. This research was made possible by NIH grant numbers: G11 HD052352, G12 RR03035, 8G12MD007583-27, U54 NS039408, UCC Pilot Project Program and Title V PPOHA grant number P031M105050 from the US Dept. of Education.

Citation Format: Kimberleve Rolon-Reyes, Miguel Mendez, David E. Rivera-Aponte, Serguei N. Skatchkov, Misty J. Eaton, Lilia Kucheryavykh. Microglia promote glioma cell migration and dispersal by activating the PLCγ1 and Pyk2 signaling cascade. [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 1644. doi:10.1158/1538-7445.AM2013-1644
Abstract 1645: Characterization of a novel anti-insoluble-fibrin chimeric antibody (AFCA) for cancer stromal target (CAST) therapy and diagnosis.
Authors: Hisada Yohei , Yasunaga Masahiro , Matsumura Yasuhiro
Published: August 2014

Background: A large body of clinicopathological evidence supports the conclusion that abnormal coagulation occurs in a variety of cancers. In the non-malignant condition, fibrin clots (insoluble fibrin) form only at the onset or active state of disease and subsequently disappear because of plasmin digestion or replacement with collagen within a few weeks. However, in cancer, fibrin clot formation and subsequent replacement with collagen continue for as long as cancer cells survive in the body. In this context we considered that the fibrin clot could be a pathophysiologically specific target in cancer and accordingly developed an anti-insoluble-fibrin chimeric antibody (AFCA). Here, we characterize AFCA and demonstrate its potential as a tool for drug delivery to the cancer stroma.

Method: The specificity of AFCA was evaluated by ELISA using insoluble fibrin, fibrinogen (the precursor of fibrin), soluble fibrin, and D-dimer (the degradation product of insoluble fibrin) as antigens. To compare AFCA with commercially available anti-fibrin antibodies, we performed ELISA with insoluble fibrin, fibrinogen, and D-dimer as antigens. We also conducted western blotting with native fibrinogen and denatured fibrinogen. To evaluate the accumulation of AFCA in tumors, we gave fluorescence-labeled AFCA to transgenic mice with pancreatic cancer.

Results: AFCA recognized only insoluble fibrin, not any fibrin-related proteins. AFCA had higher specificity against insoluble fibrin than did commercially available anti-fibrin antibodies. Western blotting analysis revealed that AFCA reacted with denatured fibrinogen but not native fibrinogen. AFCA, but not isotype control IgG, accumulated in the pancreatic cancer tissues.

Conclusion: These findings imply that the epitope of AFCA is a distinctive sequence that is uncovered only on the fibrin clot. Because lasting asymptomatic fibrin clot formation is a cancer-specific phenomenon, AFCA should be a useful tool for drug delivery to insoluble fibrin present around cancers.

Citation Format: Yohei Hisada, Masahiro Yasunaga, Yasuhiro Matsumura. Characterization of a novel anti-insoluble-fibrin chimeric antibody (AFCA) for cancer stromal target (CAST) therapy and diagnosis. [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 1645. doi:10.1158/1538-7445.AM2013-1645
Abstract 1646: Combining molecular target drugs to inhibit cancer-stromal cell interaction in gastric cancer.
Authors: Onoyama Mieko , Kitadai Yasuhiko , Shinagawa Kei , Yuge Ryo , Ohnishi Mayu , Tanaka Shinji , Yasui Wataru , Chayama Kazuaki
Published: August 2014

Recent studies in molecular and cellular biology have shown that tumor growth and metastasis are not determined by cancer cells alone but also by various stromal cells. Many human tumors secrete platelet-derived growth factor (PDGF) ligands, and PDGF receptors (PDGF-Rs) are expressed by various stromal cell populations, including carcinoma-associated fibroblasts (CAFs).

Tumor cells in surgical specimens and orthotopic tumors have been shown to express PDGF-B, whereas PDGF-Rβ has been shown to be expressed predominantly by stromal cells, including CAFs, pericytes, and lymphatic endothelial cells.

mTOR (mammalian target of rapamycin) is a central regulatory kinase that increases the production of proteins that stimulate key cellular processes such as cell growth and proliferation, cell metabolism, and angiogenesis. mTOR also increases the translation of hypoxia-inducible factor-1a (HIF-1a), which drives the expression of angiogenic growth factors such as vascular endothelial growth factor (VEGF), resulting in new vasculature.

In the present study, we examined the effects of PDGF-R tyrosine kinase inhibitors (imatinib, nilotinib) and mTOR inhibitor (everolimus) on tumor stroma in an orthotopic nude mouse model of human gastric cancer. Treatment with PDGF-R tyrosine kinase inhibitors did not inhibit tumor growth but did significantly decrease the stromal reaction, lymphatic invasion, lymphatic vessel area, and pericyte coverage of tumor microvessels. In contrast, treatment with mTOR inhibitor reduced tumor growth and microvessel density but did not decrease the stromal reaction. PDGF-R inhibitor and mTOR inhibitor used in combination reduced both the growth rate and stromal reaction. (control (5/5, 0.3±0.1g), nilotinib (6/6, 0.3±0.1g), everolimus (4/6, 0.08±0.02g: p<0.05), combination (1/6, 0.04g))

Target molecule-based inhibition of cancer-stromal cell interaction may hold promise as an effective anti-tumor therapy.

Citation Format: Mieko Onoyama, Yasuhiko Kitadai, Kei Shinagawa, Ryo Yuge, Mayu Ohnishi, Shinji Tanaka, Wataru Yasui, Kazuaki Chayama. Combining molecular target drugs to inhibit cancer-stromal cell interaction in gastric 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 1646. doi:10.1158/1538-7445.AM2013-1646
Abstract 1651: Regulation of omental seeding of ovarian cancer cells by SPARC.
Authors: Matthews Dylan P. , Said Neveen A.
Published: August 2014

Epithelial Ovarian cancer is the leading cause of death from malignant gynecologic tumors, as most patients have extensive late metastatic disease at the time of diagnosis. Ovarian cancer (OvCa) cells have specific predilection to metastasize to omentum, a condition known as “peritoneal ovarian carcinomatosis, POC” but rarely leave the peritoneal cavity. Despite aggressive surgical debulking and cytoreduction, recurrence of omental implants is high with poor patient survival. We have recently reported that the tumor suppressor role of matricellular glycoprotein SPARC (secreted protein acidic and rich in cysteine) in POC through differential effects on OvCa cells and their interactions with components of the peritoneal microenvironment. Given that SPARC is the first known matricellular protein involved in adipose tissues homeostasis, we sought to elucidate the potential roles of SPARC on the omental adipocyte-OvCa cells cross-talk and its impact on POC, in vivo and in vitro. Using primary omental SP−/− and SP+/+ omental stromal cells, we found that adipocyte-SPARC delayed the adipogenic differentiation of omental stromal cells. SP−/- omental adipocytes expressed significantly higher levels of adipogeneic markers and accumulated significantly less oil droplets than SP+/+. Primary SP−/− adipocytes promoted homing of ID8 murine OvCa cells to the omentum in vivo and their migration and invasion in 3D-cocultures in vitro. Mechanistic studies using human and murine adipocyte-OvCa genetically manipulated for SPARC expression in 3D co-cultures, revealed that the effect of SPARC suppressing adipocyte-OvCa cell cross-talk mediated by the effect suppressing the activation of cEBP-β, NFκB and AP-1 in OvCa cells as well as in adipocytes with subsequent decrease in chemokines/adipokines as well as fatty acid exchange between OvCa cells and adipocytes. Our data indicates the metastasis suppressor effect of SPARC in POC is mediated in part via inhibiting the key transcription factors involved in adipocytes differentiation and OvCa progression identifying SPARC as a potential inhibitor of OvCa cell omental seeding and dormancy.

Citation Format: Dylan P. Matthews, Neveen A. Said. Regulation of omental seeding of ovarian cancer cells by SPARC. [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 1651. doi:10.1158/1538-7445.AM2013-1651