bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1803: Combining anti-Ang-2/VEGF-A therapy with radio- and chemotherapy in glioma
Authors: Solecki Gergely , Osswald Matthias , Daniel Weber , Glock Malte , Ratliff Miriam , Müller Hans-Joachim , Krieter Oliver , Kienast Yvonne , Wick Wolfgang , Winkler Frank
Published: July 2017

Angiogenesis is a biological hallmark of malignant gliomas, but antiangiogenic strategies especially targeting the VEGF axis did not show striking antitumor activities in the majority of patients so far. Other pathways may be more relevant in selected tumor entities or patients. Further, it remains unresolved which antiangiogenic combination regimen with standard radio- and/or chemotherapy is most effective. Therefore, we compared the therapeutic effects of anti-VEGF-A, anti-Ang-2, and bispecific anti-Ang-2/VEGF-A antibodies, alone and in combination with radio- or temozolomide (TMZ) chemotherapy in a malignant glioma model using multi-parameter two-photon in vivo microscopy in mice. We demonstrate that anti-Ang-2/VEGF-A leads to strongest vascular changes including vascular normalization, both as monotherapy and when combined with chemotherapy. The latter combination regimen was accompanied by most effective chemotherapy-induced death of cancer cells independent of blood vessel proximity, indicative of a better distribution of TMZ throughout the tumor. Furthermore, the combination of anti-Ang-2/VEGF-A plus TMZ consistently resulted in decreased tumor cell motility, and decreased formation of resistance-associated tumor microtubes (TMs), which finally lead to best tumor growth inhibition. Remarkably, all these parameters were just reverted when radiotherapy was chosen as combination partner. In contrast, when anti-VEGF-A was combined with radiotherapy, vascular normalization was highest, and TM length, nuclear motility and tumor growth were concordantly reduced. In conclusion, while TMZ chemotherapy benefits most from combination with anti-Ang-2/VEGF-A, radiotherapy does from anti-VEGF-A. The findings imply that unexpected, even divergent effects can occur when a specific antiangiogenic therapy is added to chemo- or radiotherapy in glioma, and that uninformed combination regimens should be avoided.

Note: This abstract was not presented at the meeting.

Citation Format: Gergely Solecki, Matthias Osswald, Weber Daniel, Malte Glock, Miriam Ratliff, Hans-Joachim Müller, Oliver Krieter, Yvonne Kienast, Wolfgang Wick, Frank Winkler. Combining anti-Ang-2/VEGF-A therapy with radio- and chemotherapy in glioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1803. doi:10.1158/1538-7445.AM2017-1803
Abstract 1810: Dysregulation of myristoylated alanine-rich C kinase substrate (MARCKS): A novel therapeutic target in renal cell carcinoma
Authors: Chen Ching-Hsien , Chang Wen-Hsin , Yu Eric , Arif Muhammad S. , Wu Reen , Weiss Robert H.
Published: July 2017

Targeted therapeutics, such as those abrogating hypoxia inducible factor (HIF)/VEGF signaling, are initially effective against kidney cancer (or renal cell carcinoma, RCC); however, drug resistance frequently occurs via subsequent activation of alternative pathways. Through genome-scale integrated analysis of the HIF-α network, we identified the major protein kinase C substrate MARCKS (myristoylated alanine-rich C kinase substrate) as a potential target molecule for kidney cancer. In a screen of nephrectomy samples from 56 patients with RCC, we found that MARCKS expression and its phosphorylation are increased and positively correlate with tumor grade. Genetic and pharmacologic suppression of MARCKS in high grade RCC cell lines in vitro led to a decrease in cell proliferation and migration. We further demonstrated that higher MARCKS expression promotes growth and angiogenesis in vivo in an RCC xenograft tumor. MARCKS acted upstream of the AKT/mTOR pathway, activating HIF-target genes, notably VEGF-A. Following knockdown of MARCKS in RCC cells, the IC50 of the multi-kinase inhibitor regorafenib was reduced. Surprisingly, attenuation of MARCKS using the MPS peptide synergistically interacted with regorafenib treatment and decreased survival of kidney cancer cells through inactivation of AKT and mTOR. Our data suggest a major contribution of MARCKS to kidney cancer growth and provide an alternative therapeutic strategy of improving the efficacy of multi-kinase inhibitors.

Citation Format: Ching-Hsien Chen, Wen-Hsin Chang, Eric Yu, Muhammad S. Arif, Reen Wu, Robert H. Weiss. Dysregulation of myristoylated alanine-rich C kinase substrate (MARCKS): A novel therapeutic target in renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1810. doi:10.1158/1538-7445.AM2017-1810
Abstract 1812: In vitro and in vivo antiangiogenic activity of desacetylvinblastine monohydrazide through inhibition of VEGFR2 and Axl pathways
Authors: Chen Minfeng , Lei Xueping , Nie Qiulin , Hu Jianyang , Zhuo Zhenjian , Yiu Anita , Chen Heru , Xu Nanhui , Huang Maohua , Ye Kaihe , Bai Liangliang , Ye Wencai , Zhang Dong-Mei
Published: July 2017

Background and Objective: Tumor angiogenesis process is regulated by multiple proangiogenic pathways, such as VEGFR2 and Axl. Inhibition of VEGF/VEGFR2 signaling alone fails to block tumor neovascularization, and anti-VEGF resistance is often associated with Axl. Hence, discovery of novel agents that target multiple angiogenesis pathways is in demand. Here, we describe desacetylvinblastine monohydrazide (DAVLBH), a derivative of vinblastine (VLB) that exerts a more potent antiangiogenic effect than VLB in vitro and in vivo by inhibiting VEGFR2 and Axl pathways.

Methods: The antiangiogenic effects of DAVLBH were studied in vitro (proliferation, migration, and tube formation assays) and ex vivo (aortic ring assay). In vitro pericyte migration to endothelial tubes was assessed using a three-dimensional co-culture assay. In vivo assay was performed in HeLa xenograft model. Western blotting, immunohistochemical and immunofluorescence assays were conducted to evaluate the key proteins of the VEGF/VEGFR2 and Gas6/Axl pathways.

Results: DAVLBH (1 nM) inhibited VEGF- and Gas6-induced angiogenesis in vitro. At 0.75 μmol/kg, DAVLBH significantly delayed tumor growth and reduced vascular density in vivo, which was associated with the inactivation of VEGF/VEGFR2 and Gas6/Axl signalling pathways. DAVLBH blocked the compensatory upregulation of Axl in response to bevacizumab treatment in HUVECs. DAVLBH also suppressed the recruitment of pericytes to well-established endothelial tubes and reduced pericyte coverage in vivo, which was accompanied by inhibition of Axl.

Conclusions: DAVLBH potently inhibited angiogenesis-mediated tumor growth by blocking the activation of VEGF/VEGFR2 and Gas6/Axl pathways. DAVLBH might serve as a promising antiangiogenic agent for cancer therapy.

Note: This abstract was not presented at the meeting.

Citation Format: Minfeng Chen, Xueping Lei, Qiulin Nie, Jianyang Hu, Zhenjian Zhuo, Anita Yiu, Heru Chen, Nanhui Xu, Maohua Huang, Kaihe Ye, Liangliang Bai, Wencai Ye, Dong-Mei Zhang. In vitro and in vivo antiangiogenic activity of desacetylvinblastine monohydrazide through inhibition of VEGFR2 and Axl pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1812. doi:10.1158/1538-7445.AM2017-1812
Abstract 1815: Extracellular vesicles derived from lung cancer cell may contribute to the formation of malignant pleural effusion via activating Stat3/Tissue factor/VEGF
Authors: Lin Chien-Chung , Huang Wei-Lun , Su Wu-Chou
Published: July 2017

Malignant pleural effusion (MPE) is a poor prognostic sign for patients with non-small-cell lung cancer (NSCLC) and is noted around 15% at the time of initial diagnosis and 50% in their later courses. Previously, we have shown that IL-6/Stat3/tissue factor (TF)/VEGF pathway enhances lung cancer angiogenesis, metastasis and the generation of MPE. In the current study, we demonstrated that EVs derived from lung cancer cells may also contribute MPE formation. We first used ultra-filtration (UF) method as well as the classical ultra-centrifugation (UC) method to isolate EV from lung cancer cell line and demonstrated that the EVs isolated by UF method were characteristic with exosome marker in Western Blotting (Alix, CD63, and Tsg101), morphology at Transmission electron microscopy (TEM) and size in Nanosight system (30 to 150 nm). After labeling these EVs with the green fluorescent dye Cellvue, EVs uptake in AS2 cell can be imaged by the fluorescent microscopy. After the EV uptake of recipient cell, the oncogenic signal such as Stat3 and AKT were activated with increasing phosphorylation at Western blotting comparing to those adding serum free medium. Using the ELISA method, we found the cargos in these EV were enriched in IL6. We also found these cargo derived from EVs, containing high VEGF and TF, induced more prominent extravasation than serum free medium did in Miles assays. In clinical sample, we also isolated cancer cell from MPE and the purity of the cells was confirmed by immunofluorescent staining with specific TTF-1expression of lung adenocarcinoma. After short-term culture, EV from MPE cancer cell have the characteristic of exosome marker and induced more prominent extravasation than serum free medium as EV from AS2 cell did. And we also isolated EV direct from MPE and pleural effusion from congestive heart failure (CHF) patients. And we found EV derived from MPE induced more prominent extravasation than those derived from CHF in Miles assays. In conclusion, we demonstrated the UF is clinical assessable method for isolating EV from body fluid and EV derived from lung cancer cell and MPE may contribute oncogenic and angiogenic signal transmission accelerating the formation of MPE.

Citation Format: Chien-Chung Lin, Wei-Lun Huang, Wu-Chou Su. Extracellular vesicles derived from lung cancer cell may contribute to the formation of malignant pleural effusion via activating Stat3/Tissue factor/VEGF [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1815. doi:10.1158/1538-7445.AM2017-1815
Abstract 1819: Reprogramming the tumor microenvironment by targeting endothelial DNA repair
Authors: Espinosa-Diez Cristina , Wilson RaeAnna , Ruhl Rebecca , Kanner Nathan , Chatterjee Namita , Hudson Clay , Anand Sudarshan , Rana Shushan
Published: July 2017

The tumor microenvironment (TME) plays a critical role in orchestrating immune infiltration, tumor progression and response to therapeutics. Therefore, strategies to manipulate the TME including cytokine gene therapy, antibodies to reverse macrophage polarization etc are under active investigation. We have identified an alternative approach to target the TME by disrupting DNA repair in the tumor endothelial cells (ECs). We discovered a seven-microRNA (miR) signature induced in ECs in in vitro and in vivo by oxidative stress and DNA damage. The top miR candidate in this signature, miR-103 altered the TME by inducing DNA damage in ECs, eliciting type I interferons, upregulating immune costimulatory receptors and decreasing PD-L1 expressing tumor associated macrophages and granulocytes. Moreover, miR-103 treatment had a paracrine effect on the tumors and upregulated Fas and TRAIL receptors. Mechanistically, these functions of miR-103 were largely due to its downregulation of the three prime exonuclease TREX1. Local, systemic or vascular targeted delivery of miR-103 decreased both angiogenesis and tumor burden in multiple mouse tumor models.

Complementary to the role of miR-103, two additional miRs in the miR signature, miRs 494 and 99b each induced senescence in the vasculature by downregulating the Mre11a-Rad50-NBN (MRN) complex. Ectopic expression of miRs 494 or 99b decreased telomerase activity, increase p21 levels with a concomitant decrease in pRb levels. Vascular targeted delivery of miR-494 decreased angiogenesis in vivo whereas systemic delivery decreased tumor growth. Interestingly, both miR mimics and the MRN knockdowns induced the transcription of a number of senescence associated genes including CD44. Taken together these data suggest that miRs 494 and 99b targeting of the MRN complex induces senescence.

The MRN complex interacts with the ATM kinase, histone H2AX and TREX1 suggesting that the miRs we identified disrupt critical nodes of a DNA Damage Response (DDR) network. Our findings reveal a complex, miR mediated cross talk between EC DNA damage pathways, the TME and tumor cells. These interactions can be exploited for designing therapies that synergize with tumor cell killing to enhance host anti-tumor responses.

Citation Format: Cristina Espinosa-Diez, RaeAnna Wilson, Rebecca Ruhl, Nathan Kanner, Namita Chatterjee, Clay Hudson, Sudarshan Anand, Shushan Rana. Reprogramming the tumor microenvironment by targeting endothelial DNA repair [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1819. doi:10.1158/1538-7445.AM2017-1819
Abstract 1828: Are callipers obsolute? A novel 3D scanning technology to measure subcutaneous tumor volume
Authors: Wilson Zena , Delgado Juan , Davies Michael , Whiteley Rebecca , Hare Jennifer , Rahi Amar , Marshall Stephen , Smith Andrew , Atkinson Stephen , Ralli Jarno , Zabair Adeala , Zabair Adeala , Kendrew Jane
Published: July 2017

Most preclinical oncology studies (xenograft, PDX, GEMMS) involve monitoring tumour growth rates, measuring them with callipers, and calculating the volume. Volume is calculated from the width and the length to estimate a 3D volume and is directly used to assess treatment efficacy. Although this technique is useful, it is unable to accurately assess non-uniformly shaped or very small tumours and introduces a systematic bias by assuming that tumours present with spheroid shape. Furthermore callipers do not inform of the tumour condition, which is dependent upon a visual estimation.

Here we describe the development and validation of a 3D scanner as an alternative method to callipers to monitor tumour progression in rodents. The resulting 3D scanner solution made up of hardware and software, has the potential to impact on the 3Rs guiding principles underpinning the humane use of animals in oncology research. The 3Rs benefits identified are primarily through reduction of animals via improved data accuracy allowing reduction in group sizes or the ability to include irregularly shaped tumours to test. In addition the scanner system described will make it possible to record tumour measurements in a rapid, minimally invasive, morphology-independent, and human-bias-free way, removing interoperator variability. This photo-based technique captures external symptoms of redness, paleness, ulceration of tumours, etc., which could ultimately be used to detect early toxicities of compounds or determine scales of animal welfare.

We describe the development and early validation of the scanner system within our laboratories. Using the 3D scanner alongside tumour callipers to monitor tumour growth of Oncology tumour studies we demonstrated that we can accurately measure tumour size parameters (length, width and volume) in multiple mouse strains and across a range of tumour models. 3D scanning tumour data is comparable to tumour measures generated from tumour callipers If successful the introduction of this system to replace tumour callipers could have a large impact for groups running oncology in-vivo tumour studies.

Citation Format: Zena Wilson, Juan Delgado, Michael Davies, Rebecca Whiteley, Jennifer Hare, Amar Rahi, Stephen Marshall, Andrew Smith, Stephen Atkinson, Jarno Ralli, Adeala Zabair, Adeala Zabair, Jane Kendrew. Are callipers obsolute? A novel 3D scanning technology to measure subcutaneous tumor volume [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1828. doi:10.1158/1538-7445.AM2017-1828
Abstract 1831: PET/CT imaging of interleukin-13 receptor alpha-2-targeted peptide to glioblastoma after locoregional delivery
Authors: Sattiraju Anirudh , Xuan Ang , Almaguel Frankis , Herpai Denise , Debinski Waldemar , Mintz Akiva , Sai Kiran Kumar Solingapuram
Published: July 2017

Glioblastoma (GBM) is the most aggressive and common primary malignant astrocytoma which is characterized by tumor heterogeneity, infiltrating margins. Radiotherapy, chemotherapy and experimental targeted therapies have been ineffective at meaningfully increasing patient survival. One significant shortcoming of systemically delivered therapies is their inability to cross the blood brain barrier (BBB) and access infiltrating tumor cells. Therefore, in this work, we tested the potential of locoregionally targeting GBM via IL13Rα2, which we discovered to be expressed on greater than 75% of GBMs. To accomplish this, we intracranially infused copper-64 (64Cu) radiolabeled IL13Rα2 specific peptide Pep-1L, previously developed by Pandya et al., into mice bearing IL13Rα2 expressing orthotopic GBMs. Small animal micro PET/CT imaging showed ~2-fold greater tumor specific localization and lower volume of distribution of 64Cu-Pep-1L within brains of mice. Post-PET biodistribution study showed greater retention of 64Cu-Pep-1L 4 hours (%ID/g = 20.85± 0.65) and 24 hours (%ID/g = 14.65± 0.30) post infusion when compared to similarly infused 64Cu radiolabeled scrambled control peptide 4 hours (%ID/g = 10.73± 2.02) and 24 hours (%ID/g = 5.97± 1.47) post infusion. These results demonstrate that Pep-1L efficiently targets IL13Rα2 expressing GBMs in vivo upon loco-regional delivery and can effectively deliver potential therapeutic agents to GBM tumors while sparing normal brain.

This work was supported by the American Cancer Society Mentored Research Scholar grant # 124443-MRSG-13-121-01-CDD (Mintz), 1R01CA179072-01A1 (Mintz), P30 CA012197 (Pasche), R01CA07414519 (Debinski) and the Translational Imaging Program (TIP) of the Wake Forest CTSA (UL1TR001420).

Citation Format: Anirudh Sattiraju, Ang Xuan, Frankis Almaguel, Denise Herpai, Waldemar Debinski, Akiva Mintz, Kiran Kumar Solingapuram Sai. PET/CT imaging of interleukin-13 receptor alpha-2-targeted peptide to glioblastoma after locoregional delivery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1831. doi:10.1158/1538-7445.AM2017-1831
Abstract 1837: High Ras activity promotes neoplasia in pancreatic ductal cells
Authors: Singh Kanchan , Pruski Melissa A. , Dar Wasim A. , Polireddy Kishore , Bynon John S. , Younes Mamoun , Maitra Anirban , Logsdon Craig D. , Bailey Jennifer M.
Published: July 2017

Pancreatic ductal adenocarcinoma (PDAC) remains a highly lethal malignancy and is the third leading cause of cancer-related deaths in the United States. Next generation sequencing efforts have revealed KRAS mutations occur in over 99% of pancreatic ductal adenocarcinomas and are thought to be the initiating mutation for pancreatic cancer. Despite the high prevalence of KRAS mutations in pancreatic cancer patients, murine models expressing endogenous levels of mutant Kras in pancreatic cells do not develop PDAC. A number of publications have revealed that acinar cells are more sensitive to early neoplastic lesion formation in the presence of Kras mutations than ductal cells. In these models, a second genetic hit (loss of tumor suppressor TP53, SMAD4 or CDKN) is required for the development of invasive PDAC, including transforming pancreatic ductal cells. Recent data has revealed a novel role for high levels of Ras activity in acinar cells in the tranformation of this cell type to PDAC (Ji, B., 2009). We hypothesized based on other preliminary data that high levels of Ras activity would transform ductal cells. To test this hypothesis, we studied the role of constitutively active high levels of Kras activity in acinar and ductal cells by expressing an inducible KrasG12V allele in Ptf1a:CreERT2 (acinar cell specific) and Hnf1b:CreERT2 (ductal cell specific) mice. Similar to previously published work, high levels of Ras activity in acinar cells resulted in PDAC and a remarkably low survival time of 10 days after mice were injected with tamoxifen. Mice with elevated Ras activity in ductal cells needed to be euthanized two weeks after tamoxifen injection and manifested a cancer cachexic phenotype. Histological analysis of these mice revealed high grade oncocytic intraductal neoplasia and the phenotype extended throughout the pancreatobiliary ducts. Western blot analysis of human PDAC and Cholangiocarcinoma tissue revealed similar levels of Ras activity to our murine models, confirming human equivalent high Ras expression to both animal models. Oncocytic tumors are characterized by an abundance of mitochondria. Thus, we are currently studying metabolic alterations and mitochondria accumulation in ductal and acinar cells expressing high Ras activity. Our goal is to determine how mitochondrial alterations play a role in progression or inhibition of tumors in ductal vs acinar cells.

Citation Format: Kanchan Singh, Melissa A. Pruski, Wasim A. Dar, Kishore Polireddy, John S. Bynon, Mamoun Younes, Anirban Maitra, Craig D. Logsdon, Jennifer M. Bailey. High Ras activity promotes neoplasia in pancreatic ductal cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1837. doi:10.1158/1538-7445.AM2017-1837
Abstract 1866: Ultrahigh resolution optical coherence microscopy for label-free imaging of human cervical lesions ex vivo
Authors: Zeng Xianxu , Li Canyu , Wang Xiaofang , Jerwick Jason , Xu Tao , Ning Yuan , Zhang Linlin , Ma Yutao , Zhang Xiaoan , Zhou Chao
Published: July 2017

Cervical cancer remains the fourth most common cause of cancer and the fourth leading cause of cancer death in women worldwide. In contrast to the decreasing prevalence in developed countries, incidence of cervical cancer remains high in developing countries, such as China, India and Chile. Effective screening of cervical cancer is crucial for early diagnosis of the disease. Ultrahigh resolution optical coherence microscopy (UHR OCM) is an emerging label free imaging technique that can be used to obtain three dimensional (3D) “optical biopsy” of biological samples with cellular resolution up to 2 mm beneath the tissue surface. The goal of this study is to assess the diagnostic value of UHR OCM for human cervical precancer and cancer screening and identify characteristic features in UHR OCM images that correlate with the gold standard histopathology. Six hundred and fifty eight (658) fresh cervical tissues from cervical biopsy, conization and panhysterectomy were obtained from 254 consenting volunteers. Multiple 3D UHR OCM images were obtained from each specimen before the sample was processed for standard histopathology evaluation. Of the 658 samples imaged, 474 had normal cervix or benign lesions, 110 had low grade squamous intraepithelial lesion (LSIL), 57 had high grade squamous intraepithelial lesions (HSIL) and 17 had invasive cancer. UHR OCM images were compared to histopathology slides obtained from the same specimens in order to identify diagnostic image features. Distinctive patterns for squamous epithelium, columnar epithelium, basement membrane, cervical inflammation and erosion, koilocytic cells, low grade and high grade squamous intraepithelial lesions and cervical cancer were clearly observed. Our results lay the foundation for future non-invasive optical evaluation of cervical tissue in vivo. This could lead to a less invasive and more effective screening strategy for cervical cancer based on OCM guided biopsies.

Citation Format: Xianxu Zeng, Canyu Li, Xiaofang Wang, Jason Jerwick, Tao Xu, Yuan Ning, Linlin Zhang, Yutao Ma, Xiaoan Zhang, Chao Zhou. Ultrahigh resolution optical coherence microscopy for label-free imaging of human cervical lesions ex vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1866. doi:10.1158/1538-7445.AM2017-1866
Abstract 1878: PDZ-RhoGEF functions as a critical signaling effector for TROY
Authors: Ding Zonghui , Roos Alison , Kloss Jean , Dhruv Harshil , Symons Marc , Tran Nhan L. , Loftus Joseph C.
Published: July 2017

Glioblastoma Multiforme (GBM) is the most common tumor of the CNS with a median survival of approximately 15 months. The highly aggressive invasion of malignant cells into the surrounding normal brain tissue renders complete surgical resection impossible, increases therapeutic resistance, and virtually assures tumor recurrence. We have previously demonstrated that TROY (TNFRSF19) has an important role in GBM invasion and resistance. TROY expression is correlated with glial tumor grade and inversely correlated with patient survival. Increased TROY expression stimulated cell migration/invasion in vitro and in vivo and confers resistance to both ionizing radiation- and temozolomide-induced apoptosis via activation of Akt and NF-κB. These data indicate that TROY stimulated migration/invasion is associated with signaling pathways that also increase survival and therapeutic resistance, however, the mechanistic basis of this signaling remains unclear. To investigate the mechanism by which TROY induces GBM cell invasion, we performed immunoprecipitation of the TROY receptor coupled with MALDI-TOF and MS/MS analysis and identified PDZ-RhoGEF as a binding partner for TROY. We validated the interaction of TROY with PDZ-RhoGEF by immunoprecipitation and western blotting, and demonstrated that PDZ-RhoGEF can exchange for both RhoA and RhoC in glioma cells and is implicated in GBM cell invasion, proliferation, and survival. In order to assess the specific role of PDZ-RhoGEF in TROY signaling, we examined the effects of PDZ-RhoGEF on TROY-mediated activation of NF-κB. While overexpression of TROY alone strongly stimulated NF-κB transcriptional activity in serum-free conditions, increased expression of PDZ-RhoGEF alone had no effect on NF-κB activity. Co-transfection of PDZ-RhoGEF with TROY synergistically promoted NF-κB activation. Mutation of the TRAF binding domain (TBD) in the cytoplasmic domain of TROY abolished its capacity to activate NF-κB, even in the presence of PDZ-RhoGEF, suggesting that the TBD is important for TROY induced NF-κB activation. Furthermore, knockdown of PDZ-RhoGEF by shRNA attenuated NF-κB activation in TROY expressing cells, blocked TROY mediated invasion of primary GBM xenograft cells and increased sensitivity to temozolomide. Collectively, these data indicate that PDZ-RhoGEF plays an important role in TROY signaling and provides insights into a potential node of vulnerability to limit GBM cell invasion and decrease therapeutic resistance.

Citation Format: Zonghui Ding, Alison Roos, Jean Kloss, Harshil Dhruv, Marc Symons, Nhan L. Tran, Joseph C. Loftus. PDZ-RhoGEF functions as a critical signaling effector for TROY [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1878. doi:10.1158/1538-7445.AM2017-1878
Abstract 1891: Annexin A2-binding S100 proteins promote proliferation and cell cycle progression of EGFR positive cancer cells
Authors: Ogama Naoko , Nagashima Ryuuichi , Tanaka Nobuyuki
Published: July 2017

(a) Aim: The purpose of this study is to clarify an unknown function of Annexin A2-binding S100A proteins in tumor growth, cell cycle progression and invasion. Annexin A2 is a membrane bound protein originally identified as v-Src substrate, and often found to be overexpressed in many types of cancers. As a multicompartmental protein that orchestrates a spectrum of dynamic membrane-related events, it is associated with actin cytoskeleton control, vesicle formation and intercellular interaction.

(b) Methods and Results: During our proteomics analysis using solid tumor cell lines, we identified both Annexin A2 and S100A proteins in cancer stem cell-rich population. We next examined S100A proteins expression in several Head and Neck cancer PDX samples. Immunohistochemical data from in vivo specimens as well as western blots showed that S100A expression abundance was well associated with poor prognosis. Annexin A2 has been indicated to be involved in malignant phenotypes such as invasion and metastasis, and Annexin A2 and S100A form (S100-Annexin A2)x2 dimers, we next examined roles of S100A proteins in cancer, by using shRNA-mediated depletion experiments.

(c) New Findings: Depletion of S100A from PDX-derived cancer cells resulted in loss of both in vivo tumor formation and invasion. Furthermore, cell cycle progression was inhibited by the depletion, suggesting its potential role in proliferation controls. Similar to the mutant Ras-driven tumors, EGFR-overexpression increased S100A gene expresssion.

(d) Conclusion: These results strongly suggest that increases in S100A plays significant roles in cancer cell proliferation and invasion.

Note: This abstract was not presented at the meeting.

Citation Format: Naoko Ogama, Ryuuichi Nagashima, Nobuyuki Tanaka. Annexin A2-binding S100 proteins promote proliferation and cell cycle progression of EGFR positive cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1891. doi:10.1158/1538-7445.AM2017-1891
Abstract 1892: Small molecule NSC59984 prevents cancer cell migration and invasion
Authors: Zhang Shengliang , Wagner Jessica , Zhou Lanlan , El-Deiry Wafik S.
Published: July 2017

Tumor metastasis is one of the main mechanisms leading to mortality of cancer patients. Searching for new therapeutic strategies to prevent tumor metastasis is one of the anti-tumor approaches in cancer therapy. Cell migration and invasion are crucial initial steps in the complex process and progression of tumor metastasis. Mutant p53 has been known as an important factor associated to cancer cell migration. Therefore, targeting mutant p53 is a potential strategy to avoid tumor metastatic spread. We previously reported an anti-tumor small molecule, NSC59984 which degrades mutant p53 and restores p53 pathway via activation of p73. Here we demonstrate that NSC59984 inhibits cancer cell migration and invasion. Treatment with NSC59984 partially attenuates cell migration in various cancer cells based on the scratch assay and Boyden chamber assay. The effect of NSC59984 on cell migration is partially blocked by knockdown of p73, a member of the p53 family, suggesting that restoration of p53 pathway signaling via p73 is required for NSC59984-inhibition of cell migration in cancer cells. We found that NSC59984 induces ERK2-dependent p53 restoration and mutant p53 degradation. To examine the role of ERK2 in the cell migration, ERK2 signaling is blocked by U0126 treatment in cancer cells. The effect of NSC59984 on cancer cell migration is partially inhibited by U0126 treatment, suggesting that NSC59984 inhibits cancer cell migration partially dependent on ERK1/2 pathway signaling in cancer cells. In addition, the gelatin degradation assay shows that NSC59984 inhibits cancer cell invasion. Consistent to the in vitro assays, NSC59984 decreases tumor invasion of xenograft tumors as compared to the xenograft tumors treated with DMSO as control. Our results, taken together, suggest that NSC59984 not only suppresses tumor growth, but also inhibits cancer cell migration and invasion. Our findings provide the potential administration of p53-restoring compound NSC59984 in cancer therapy by blocking tumor metastasis.

Citation Format: Shengliang Zhang, Jessica Wagner, Lanlan Zhou, Wafik S. El-Deiry. Small molecule NSC59984 prevents cancer cell migration and invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1892. doi:10.1158/1538-7445.AM2017-1892
Abstract 1898: MEG3 as a competing endogenous RNA binds with miR-27a to promote PHLPP2 protein translation, and consequently impairs bladder cancer invasion
Authors: Huang Chao , Liao Xin , Li Jingxia , Huang Chuanshu
Published: July 2017

Muscle invasion and metastasis of bladder cancer dramatically decrease survival rate, compared to 77% of 5 year survival rate of all bladder cancer, metastasis bladder cancer is only 5%. This truth implies that one of the therapeutic kernels of bladder cancer is to decrease, even block, its invasiveness. In the current study, we find that MEG3 is significantly downregulate in human muscle-invasive bladder cancer tissues compared with non-muscle invasive tissues. We also demonstrate the MEG3 strongly inhibits human bladder cancer cell invasiveness by acting as a ceRNA. We find that MEG3 competes with PHLPP2 mRNA for miR-27a binding, which leads to a decreased miR-27a activity, and subsequently reducing the translation inhibition of miR-27a to PHLPP2. The upregulated PHLPP2 decreases the c-Jun phosphorylation at Ser63/73, and in turn inhibits the c-Myc transcription and its mediated invasiveness of the bladder cancer cells. This is the first demonstration that MEG3 inhibits the invasiveness of human bladder cancer cells via negative regulation of c-Myc as a ceRNA. These findings not only provides a novel insight into understanding the mechanisms behind the MEG3 inhibiting the cancer cell invasion, but also reveals a potential targets for invasive bladder cancer prevention and therapy.

Citation Format: Chao Huang, Xin Liao, Jingxia Li, Chuanshu Huang. MEG3 as a competing endogenous RNA binds with miR-27a to promote PHLPP2 protein translation, and consequently impairs bladder cancer invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1898. doi:10.1158/1538-7445.AM2017-1898
Abstract 1913: SOX9 regulates cancer stem-like cells in non-small cell lung cancer
Authors: Voronkova Maria , Luanpitpong Sudjit , Riedel Heimo , Rojanasakul Yon
Published: July 2017

Lung cancer is the leading cause of cancer death worldwide in both men and women, every year killing more people than breast, prostate and colon cancers combined. Drug/radiation resistance and tumor relapse contribute to low patient survival, which has largely been attributed to the acquisition of stem-like cells (CSCs), or tumor initiating cells. Moreover, conventional therapies are not effective against CSCs. This signifies the need to identify mechanisms of CSC regulation which could lead to the discovery of CSC-specific drug targets and the development of more effective drug therapies. An embryonic transcription factor SOX9 has been implicated in CSC regulation in a number of cancer types, but little is known about its role in non-small cell lung cancer (NSCLC). We demonstrated that high SOX9 expression correlates with poor survival in lung cancer patients by analyzing The Cancer Genome Atlas (TCGA) data. We hypothesized that SOX9 plays a key role in tumor progression and chemoresistance by upregulating CSCs in NSCLC. To test this hypothesis, SOX9 expression was stably or transiently knocked down in NSCLC cells, and their effects on CSC formation and biomarkers, and on drug resistance were investigated. Our results showed that SOX9 knockdown decreased the number of tumor spheres in NSCLC cell lines. Furthermore, SOX9 knockdown downregulated the expression and activity of stem cell marker ALDH1A1 as determined by Western blot and flow cytometric assays. Consistent with this finding, overexpression of SOX9 in non-cancer lung epithelial Beas-2B cells elevated tumor sphere formation, supporting the role of SOX9 in CSC regulation. Analysis of the soft agar colony formation assay further showed that SOX9 knockdown inhibited anchorage-independent growth in NSCLC cells. More importantly, SOX9 knockdown resulted in an increased sensitivity of the cells to a chemotherapeutic drug cisplatin, whereas SOX9 overexpression decreased it. Together, our results indicate an important role of SOX9 in the regulation of CSCs in NSCLC and suggest its potential utility as a drug target for chemoresistant lung cancer.

Citation Format: Maria Voronkova, Sudjit Luanpitpong, Heimo Riedel, Yon Rojanasakul. SOX9 regulates cancer stem-like cells in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1913. doi:10.1158/1538-7445.AM2017-1913
Abstract 1925: Modeling physiologic microenvironments in three-dimensional microtumors facilitates brain tumor initiating cell maintenance
Authors: Gilbert Ashley , Walker Kiera , Tran Anh , Gillespie Yancey , Singh Raj , Hjelmeland Anita B.
Published: July 2017

Development of effective novel anti-tumor treatments will require improved in vitro models that incorporate physiologic microenvironments and maintain intratumoral heterogeneity including tumor initiating cells. Brain tumor initiating cells (BTIC) are a target for cancer therapy because they are highly tumorigenic and contribute to tumor angiogenesis, invasion, and therapeutic resistance. Current leading studies rely on BTIC isolation from patient-derived xenografts followed by propagation as neurospheres. As this process is expensive and time-consuming, we determined whether three-dimensional microtumors were an alternative in vitro method for modeling tumor growth via BITC maintenance and/or enrichment. Brain tumor cells were grown as neurospheres or as microtumors produced using a human-derived biomatrix HuBiogelTM and maintained with physiologically relevant microenvironments. Percentages of BITCs were determined based on cell surface marker expression (CD133), label retention (carboxyfluorescein succinimidyl ester; CFSE), and tumorsphere formation capacity. Our data demonstrate that expansion of brain tumor cells as hypoxic and nutrient restricted microtumors significantly increased the percentage of both CD133+ and CFSE+ cells. We further demonstrate that BTIC-marker positive cells isolated from microtumors maintain neurosphere formation capacity in the in vitro limiting dilution assay and tumorigenic potential in vivo. These data demonstrate that microtumors can be a useful three-dimensional biological model for the study of BTIC maintenance and targeting.

Citation Format: Ashley Gilbert, Kiera Walker, Anh Tran, Yancey Gillespie, Raj Singh, Anita B. Hjelmeland. Modeling physiologic microenvironments in three-dimensional microtumors facilitates brain tumor initiating cell maintenance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1925. doi:10.1158/1538-7445.AM2017-1925
Abstract 1931: The first circulating tumor cell detection technique from frozen PBMCs
Authors: Li Heming , Batth Izhar Singh , Xia Xueqing , Hsu Frank J. , Somaiah Neeta , Torres Keila Enitt , Wang Ruoyu , Li Shulin
Published: July 2017

Circulating tumor cells (CTCs) enter the vasculature or lymphatic system after shedding from the primary tumor. CTCs may serve as “seed” cells for tumor metastasis. The utility of CTCs in clinical application is not fully investigated, partly due to the necessity for fresh blood samples and the lack of CTC specific antibody. Of note, there is no CTC detection tool for pediatric tumors. To overcome these drawbacks, we developed a protocol for CTC capture from frozen peripheral blood mononuclear cells (PBMCs) based on the cell-surface vimentin (CSV) antibody 84-1. CSV is a unique marker compared to others such as EpCAM because it is only specific to tumor cells and has been demonstrated to be a viable target for isolation and enumeration of CTCs across tumor types. In this study, we report the first CTC isolation technology from frozen PBMCs of osteosarcoma patients using the CSV antibody 84-1. The CTCs captured using this new protocol were validated by single cell gene sequencing analysis and mesenchymal marker α-SMA staining. Moreover, spiking analysis was also performed to ensure the specificity and sensitivity of this unique CTC capture analysis. Linear regression analysis yielded a positive correlation between the number of detected sarcoma cells and the number of sarcoma cells spiked in both fresh and frozen PBMCs. In summary, our results demonstrate for the first time, a technology to specifically detect and isolate mesenchymal origin CTCs from frozen PBMCs. This technology can be easily expanded to other types of cancers including pediatric tumors because the antibody used is universal and CTC specific. Such a technology will boost the feasibility and utility of CTC-based diagnosis and therapeutic treatment monitoring in a centralized laboratory.

Citation Format: Heming Li, Izhar Singh Batth, Xueqing Xia, Frank J. Hsu, Neeta Somaiah, Keila Enitt Torres, Ruoyu Wang, Shulin Li. The first circulating tumor cell detection technique from frozen PBMCs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1931. doi:10.1158/1538-7445.AM2017-1931
Abstract 1933: Discovery of first-in-class small molecule CD99 inhibitors for targeted therapy of Ewing sarcoma
Authors: Celik Haydar , Sciandra Marika , Flashner Bess , Gelmez Elif , Kayraklıoğlu Neslihan , Allegakoen David V. , Petro Jeff R. , Conn Erin J. , Hour Sarah , Han Jenny , Oktay Lalehan , Tiwari Purushottam B. , Hayran Mutlu , Manara Maria Cristina , Toretsky Jeffrey A. , Scotlandi Katia , Uren Aykut
Published: July 2017

Ewing sarcoma (ES) is an aggressive bone and soft tissue malignancy that affects predominantly children and adolescents with a high propensity to metastasize and poor prognosis. CD99 is a transmembrane cell surface protein that is highly expressed on ES cells, and routinely used as a marker for histological diagnosis of ES. We screened small molecule libraries for their binding to recombinant CD99 protein and subsequent selective inhibition of ES cell growth. We identified two structurally similar FDA-approved nucleoside analogues, clofarabine and cladribine that selectively inhibited the growth of ES cells in a panel of 14 ES vs. 28 non-ES cell lines. A significant negative correlation was found in human cell lines between CD99 expression and IC50 values for clofarabine and cladribine. Both drugs inhibited CD99 dimerization and its interaction with downstream signaling components cyclophilin A and PKA-RIIα as well as led to reduced ROCK2 protein expression and migration in ES cells. A membrane-impermeable analog of clofarabine showed similar cytotoxicity in ES cells, suggesting that it can function through inhibiting CD99 alone without any effect on DNA metabolism. Clofarabine and cladribine led to a significant increase in hypodiploid DNA content of ES cells, which was diminished by suppression of CD99 expression. Both drugs drastically inhibited anchorage-independent growth of ES cells, but clofarabine was more effective in inhibiting ES xenografts. Finally, the screening of a set of chemotherapy drugs revealed a synergy for the combination of anti-CD99 drugs and dasatinib in ES cells, which may translate into increased survival and reduced toxicity. Overall, our findings suggest that clofarabine is a good candidate for early phase clinical trials in children with ES.

Citation Format: Haydar Celik, Marika Sciandra, Bess Flashner, Elif Gelmez, Neslihan Kayraklıoğlu, David V. Allegakoen, Jeff R. Petro, Erin J. Conn, Sarah Hour, Jenny Han, Lalehan Oktay, Purushottam B. Tiwari, Mutlu Hayran, Maria Cristina Manara, Jeffrey A. Toretsky, Katia Scotlandi, Aykut Uren. Discovery of first-in-class small molecule CD99 inhibitors for targeted therapy of Ewing sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1933. doi:10.1158/1538-7445.AM2017-1933
Abstract 1949: Salvage pathway enzyme HPRT as a molecular marker for Burkitt’s Lymphoma
Authors: Townsend Michelle H. , Lattin John Ellis , Anderson Michael D. , Felsted Abigail , Velazquez Edwin , Weagel Evita , Robison Richard , O'Neill Kim L.
Published: July 2017

The aim of this study is to investigate the potential of Hypoxanthine phosphoribosyltransferase (HPRT) as a surface biomarker and target for future immunotherapies in Burkitt’s B-cell Lymphoma. B-cell malignancies are the most common type of childhood cancer. Development of immunotherapies could improve current treatment. HPRT is a cytosolic transferase involved in nucleotide production via the purine salvage pathway. Altered expression of TK1, a salvage pathway enzyme, is an indicator of prognosis and diagnosis in multiple cancer types due to active proliferation of cells and the resulting elevated nucleotide demand. It has previously been reported that some salvage pathway enzymes are found on the surface of Burkitt’s Lymphoma cells. HPRT presence on the surface of this B-cell lymphoma subtype could provide a target for adoptive cell transfer and other immunotherapies. The potential surface presentation of HPRT was assessed using flow cytometry, scanning electron microscopy (SEM), and cytoplasmic staining on both healthy lymphocytes and Burkitt’s Lymphoma cells (Raji). Flow cytometry experiments with HPRT antibodies and fluorescent-labeled secondary antibodies show that Raji cells exhibit an 81.4% (p-value .0001) positive fluorescence shift when compared to IgG controls (1.5%). Healthy lymphocytes had a fluorescence shift of 2.38% (p-value .9787). The presence of HPRT on the surface of both Raji cells and healthy lymphocytes was further confirmed using gold-labeled antibodies. Utilizing a scanning electron microscope, the presence of the protein on the surface was evaluated and quantified via increases in gold weight percentage of the sample. When treated with antibodies against HPRT, there was a significant increase in gold binding along with an increase in gold weight percentage. These results suggest a direct relationship between HPRT and the surface of Burkitt’s lymphoma cells, indicating HPRT as a potential target for future immunotherapeutic treatment in Burkitt’s B-cell lymphoma pediatric patients.

Citation Format: Michelle H. Townsend, John Ellis Lattin, Michael D. Anderson, Abigail Felsted, Edwin Velazquez, Evita Weagel, Richard Robison, Kim L. O'Neill. Salvage pathway enzyme HPRT as a molecular marker for Burkitt’s Lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1949. doi:10.1158/1538-7445.AM2017-1949